BEZ235 enhances antitumor efficacy of HPG-PCL-PTX in HCC by suppressed the PI3K/Akt/mTOR and MAPK pathway

BEZ235 enhances antitumor efficacy of HPG-PCL-PTX in HCC by suppressed the PI3K/Akt/mTOR and MAPK pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article BEZ235 enhances antitumor efficacy of HPG-PCL-PTX in HCC by suppressed the PI3K/Akt/mTOR and MAPK pathway Xueke Liu, Yingping wu, We Xu, Lele Li, Xiaoping Xia, Xiaolong Tang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1793410/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hepatocellular carcinoma (HCC) is the sixth most common cancer in the global. It could be a potential resistant mechanism that paclitaxel activated the PI3K/mTOR and MAPK pathway in HCC cells. So inhibiting the abnormal activation of two pathway, which may a promising targeted therapy strategy. MTT and clone formation assays were used to measure cells proliferation activity; cell cycle distribution and apoptosis were measured by flow cytometry; western blot assessed phosphorylation level of proteins associated with cell cycle, apoptosis, PI3K/mTOR and MAPK pathway; Student t test or one-way ANOVA to measure significant differences between the means.BEZ235 combined with HPG-PCL-PTX blocked the activation of PI3K/mTOR and MAPK pathway in HCC cells, effectively inhibited cells proliferation and arrested the cell cycle at the G2/M phase. The most important is that the combination of two drugs caused mitochondrial membrane potential change, induced cytochrome C release and bind to APAF-1 to initiate the caspase-9 apoptosis program, promoted HCC cell apoptosis. HPG-PCL-PTX/BEZ235 enhanced antitumor effectiveness in HepG2R cell xenograft mouse models. In vivo study suggested that BEZ235 combined with HPG-PCL-PTX enhanced effects of HPG-PCL-PTX through inhibited the PI3K/Akt/mTOR and MAPK pathway in HCC cells, supressed cells proliferation, arrested the cell cycle in a G2/M phase and promoted apoptosis. Antitumor effects of BEZ235 and HPG-PCL-PTX were also confirmed in mice bearing HepG2R tumors. Taken together, the results of this study describe a promising strategy using PTX and BEZ235 in a nanoparticle formulation for treatment of PTX-resistant HCC. Cell Cycle & Proliferation Cell Communication and Signaling Cell Migration and Cell Adhesion Cell Survival and Cell Death Drug Delivery HPG-PCL-PTX BEZ235 PI3K/Akt/mTOR HCC resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Hepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumors worldwide [ 1 – 2 ] . Like many other cancers, acquired resistance is the biggest hurdles for effective treatments [ 3 ] . Molecular targeted therapy for the signaling pathways that induce to resistance of HCC is a novel and promising treatment approach. Identification of the key target molecules and signaling pathways responsible is crucial in order to develop effective targeted therapies against HCC [ 4 – 5 ] . Paclitaxel (PTX) is an approved chemotherapeutic in the UK for breast, lung, prostate and other types of solid tumors [ 6 ] , but the low solubility hinders its application in the clinic. In addition, PTX cause some side effects such as hypersensitivity reactions, myelosuppression, and peripheral neuropathy [ 7 ] . This makes the nano-delivery system a useful tool to assist with PTX solubility and delivery. Hyperbranched polyglycidyl ethers (HPG) have a wide range of applications in the biomedical field due to their low viscosity, long in vivo circulation time and large number of terminal modifiable groups [ 8 – 9 ] . In this study, we first synthesize an amphiphilic polymer PCL-HPG using a degradable multi-armed polycaprolactone (PCL) as the core and a bisulfide bonded HPG as the arm, which uses a hydrophobic core PCL to load hydrophobic drugs, allowing the drug-loaded micelles to be stable in vivo and to achieve long circulation times, and increases the sensitivity of tumour cells to chemotherapeutic drugs to a certain extent [ 10 – 12 ] . The phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) signaling pathways play a central role for many tumor types in tumor cell proliferation, motility, invasion, metabolism and survival [ 13 ] . Approximately 10% of HCC patients carry a PIK3CA mutation in HCC [ 14 – 15 ] , these mutations can activate PI3K and can lead to the activation of Akt via phosphorylation at Thr308 through PDK1 or/and at Ser473 through the mTOR associated with Rictor (the mTORC2 complex) [ 16 ] . Activated Akt regulates key downstream effectors including mTOR associated with Raptor (mTORC1 complex), p70S6 kinase and 4E-BP1 [ 17 ] . Rapamycin and its analogous inhibit mTORC1 signaling and activate Akt signaling via mTORC2 related negative feedback loop. Abnormal activation of Akt and mTOR can reduce the effectiveness of anti-tumor drugs through promoting cellular proliferation, cell-cycle progression, inhibits the expression of mitochondrial pro-apoptotic-related proteins and prevents initiation of the pro-apoptotic caspase pathway, thus promoting cell survival [ 18 ] . Interestingly, mTOR kinase is an important regulator of inducing autophagy. Activated mTOR (Akt and MAPK signaling) inhibits autophagy, while negative regulation of mTOR (AMPK and p53 signaling) promotes autophagy. mTORC1 inhibits the initiation of autophagy through the ULK complex. mTORC1 induces phosphorylation of ULK1 at Ser758, represses the interaction between ULK1 and AMPK, and inhibits the activity of the ULK1 complex by phosphorylating ATG13, inducing phosphorylation of AMBRA1 to inhibit autophagy [ 19 ] . Thus, the combined inhibition of both PI3K and mTOR might be necessary for effective treatment of cancer. NVP-BEZ235 (BEZ235), a novel dual PI3K/mTOR inhibitor, inhibits the catalytic subunit p110a of PI3K by competing at its ATP binding site and other class 1 PI3K enzymes and also inhibits the catalytic activity of mTOR [ 20 – 21 ] . It was recently reported to mediate some anti-tumor effects in experimental pancreatic cancer [ 22 ] , breast cancer [ 23 ] and gastric cancer [ 24 ] . Taken together, targeting PI3K/mTOR or MAPK signaling is a promising option. We evaluated targeting the PI3K/mTOR or MAPK pathways antitumor efficacy of BEZ235 combined with HPG-PCL-PTX in vivo and in vitro, determine more effective HCC therapeutic strategy. Our work provides ideas and directions for HCC resistance further research. Results Characterization of HPG-PCL-PTX To improve the ratio of drug concentration and effective combination in target cells and to overcome the short plasma half-life and adverse side effects of drugs, we applied HPG-PCL polymer nanoparticles to load PTX (PTX 5.0µM) to deliver PTX to HCC. From the Fig. 1 A, it can be seen that the chemical shifts of 1.3, 1.6, 2.3 and 3.99ppm are the methylene proton absorption peaks at different positions on 4-arms-PCL, respectively. The proton absorption peak at the chemical shift of 33.4ppm is attributed to the methylene and methine groups of HPG, and the absorption peak at 4.6ppm is attributed to the hydroxyl group of HPG. The successful synthesis of HPG-PCL was demonstrated. By analyzing the particle size of the nanoparticles with DLS, it could be seen that the particle size of drug-loaded nanoparticles (HPG-PCL-PTX) was approximately 80 ~ 100nm diameter (Fig. 1 B). Transmission electron microscopic images (Fig. 1 C) reveal that the HPG-PCL and HPG-PCL-PTX are mostly spherical, relatively uniform in size, and have good monodispersity. Through the peak area in HPLC figure to calculate the drug loading rate is 2.2% and 8.2%(Fig. 1 D). BEZ235 combined with HPG-PCL-PTX inhibits proliferation of HCC cells Chemotherapy normally causes severe side effects, even in combination treatments with different drugs. Nanodelivery of drugs may provide a solution to reduce these side effects by further decreasing the dosage. To achieve this, we used HPG-PCL to load PTX combined with BEZ235 to further increase their efficacy. PTX-resistant cell sublines HepG2R were established using exposure to escalating doses of PTX. Figure 2 A shows half-maximal inhibitory concentration (IC50) values. In addition, clone formation experiments showed that BEZ235 combined with HPG-PCL-PTX can efectively inhibit the proliferation of HepG2 and HepG2R cells (Fig. 2 B and 2 C). These data confirmed that HPG-PCL-PTX combined with BEZ235 has a synergistic treatment effect on HCC. Targeting of HPG-PCL-FITC on HCC cells To evaluate the uptake efficiency of nano drug (HPG-PCL-PTX) by cells, confocal microscopy was used to detect the content of tracer nanoparticle (HPG-PCL-PTX-FITC) in the HepG2 and HepG2R cell lines at defined time points, respectively. Timed visualization detected differences in cellular uptake by HPG-PCL-PTX. After incubation for 0.5 h, 1h, 2h, 4h, the endocytosis rates of HepG2 to HPG-PCL-PTX were 15.37%, 38.42%, 57.16%, 89.78%; the endocytosis rates of HepG2R to HPG-PCL-PTX were 9.64%, 17.92%, 48.57%, 85.34%. With the prolongation of time, the phagocytosis rate of HPG-PCL-PTX by hepG2 and HepG2R cells tended to be the same, both peaked at 4h(Fig. 3 ). These results indicated that the efficient endocytosis of HPG-PCL-PTX by the cells resulted in higher cellular internalization of the drug. PTX induces abnormal activation of PI3K/mTOR and MAPK pathways in HepG2 and HepG2R cells Previous studies have noted that the involvement of the PI3K/mTOR and MAPK pathways in PTX resistance, and both pathways are further activated in PTX-resistant HCC cells compared with parental cells [ 25 ] . To determine whether the resistance to PTX of HCC cells is related to activation of PI3K/mTOR and MAPK pathway, we detected the phosphorylation levels of key molecules on the pathways in HepG2 and HepG2R cells by Western blot. The results showed that the treatment of PTX on HepG2 cells caused stress activation of key molecules in PI3K/mTOR and MAPK pathways, and peaked at 24h. While, the effect of PTX on HepG2R cells led to the stable activation of key molecules in this pathway after 24h(Fig. 4 ). Thus, PTX resistance in HCC may be due to stable activation of PI3K/mTOR and MAPK signaling. BEZ235 combination of HPG-PCL-PTX significantly inhibits activation of PI3K/mTOR and MAPK pathway in HCC cells To understand the therapeutic potential of targeting these two pathways to treat HCC cells, we assessed the PI3K/mTOR/Erk phosphorylation levels after 24h of PI3K/mTOR dual inhibitor BEZ235 in HCC cells by Western blot. The results showed that the BEZ235 combined with HPG-PCL-PTX effectively inhibted the PI3K/mTOR phosphorylation in HCC cells(Fig. 5 A and 5 B). However, when BEZ235 combined with PTX or HPG-PCL-PTX, these drugs elicited higher inhibition on Erk activity in resistant cells, yet in parental cells augmented Erk activity(Fig. 5 C). These might be due to that targeting PI3K/mTOR and MAPK pathways would induce re-activation of Akt/Erk in progenitor cells, whereas after the acquisition of PTX resistance, this feedback was attenuated, making HepG2R more sensitive to combined agents. This might be a promising instruction to overcome induced chemoresistance in cancer treatment, yet our hypothesis required further investigation. BEZ235 combined with HPG-PCL-PTX induced cell cycle arrest in the G2/M phase Induction of cell cycle arrest at the G2/M phase is the most prominent consequence of microtubular stabilization by PTX. To examine whether the combination treatment with HPG-PCL-PTX and BEZ235 enhanced this effect on the cell cycle of HCC, we carried out a cell cycle analysis by fow cytometry. The results showed that the combination treatment remarkably all increased the G2/M cell population in HepG2 and HepG2R after 24 hours treatment, compared to the single drug treatments and the untreated controls (Fig. 6 A and 6 B). We also observed a increased the sub-G1 (apoptotic) cell population after 24 hours treatment, however remarkable decrease in the G1 phase. It revealed that HPG-PCL-PTX combined with BEZ235 promoted apoptosis of HCC cells. CyclinB1 and activation of the cyclin-dependent kinase 1(CDK1)/cyclinB1 complex have been shown to regulate the cell cycle transition from G2 to the M phase. Increased expression of cyclinB1 accelerates mitosis and may lead to excessive proliferation of cells [ 26 ] . Thus, we examined the expression of endogenous cyclin B1 and CDK1 by western blot. The results showed that the expression of cyclin B1 and CDK1 in hepG2R cells was significantly higher than in HepG2 cells ( Fig. 6 C ) . However, the expression of the cyclin B1 and CDK1 were reduced in HCC cell lines when treated with HPG-PCL-PTX plus BEZ235(Fig. 6 D). These data indicated that HPG-PCL-PTX and BEZ235 in combination enhanced cells arrest at G2/M phase through the down-regulation of the cyclin B1 and CDK1, inhibited cell proliferation and promoted cell apoptosis. BEZ235 combined with HPG-PCL-PTX induced apoptosis in HCC cells JC-1 assay was used to detect the mitochondrial dysfunction of the cells [ 27 ] . To investigate the effect of apoptosis induced by HPG-PCL-PTX combined with BEZ235 in HCC cells, flow cytometry analysis and JC-1 staining for mitochondrial membrane potential (ΔΨm) were performed. In flow cytometry analysis, the green/red fluorescence intensity ratio of HPG-PCL-PTX group(HepG2:27.5%, HepG2R:29.9%) was significantly higher than HPG-PCL group(HepG2:7.8%, HepG2R:4.3%), PTX group(HepG2:15.6%, HepG2R:12%), BEZ235 group(HepG2:13.4%, HepG2R:7.7%), PTX + BBEZ235 group(HepG2:17.2%, HepG2R:20.6%) and control group (HepG2:8.2%, HepG2R:5.2%) ( p < 0.05). While HPG-PCL-PTX combined with BEZ235, the green/red fluorescence intensity ratio was significantly higher than HPG-PCL-PTX group (HepG2: 70.1%, HepG2R: 49.6%)(Fig. 7 A). JC-1 staining showed the green fuorescence intensity of HCC cells treated with HPG-PCL-PTX monotherapy increased only slightly. Interestingly, when combining with BEZ235, the green fuorescence intensity was signifcantly enhanced(Fig. 7 C). These results suggested that HPG-PCL-PTX combined with BEZ235 induces apoptosis of HCC cells by changing mitochondrial membrane potential. PTX-mediated mitotic arrest causes apoptosis, however, resistance to apoptosis frequently arises during long-term treatment [ 28 ] . So, We assayed the effect of PTX and BEZ235 combination treatment on cell death using the Annexin V FITC/PI apoptosis kit. In flow cytometry analysis, HPG-PCL-PTX (HepG2:8%, HepG2R:6.1%) caused some apoptotic cell death and slight early apoptosis after 24h treatment, was sightly higher than HPG-PCL group(HepG2:2.6%, HepG2R:2.6%), PTX + BEZ235 group(HepG2:5.1%, HepG2R:5.2%), PTX group(HepG2:3.0%, HepG2R:2.7%), BEZ235 group(HepG2:3.4%, HepG2R:3%) and control group(HepG2:3.3%, HepG2R:2.7%). Whereas the combinatorial application of HPG-PCL-PTX and BEZ235 produced a greater effect, inducring a higher apoptotic rate in HCC cells(HepG2:15.5%, HepG2R:15%). The apoptotic rate of HepG2 cells were slighterly higher than in HepG2R cells(Fig. 7 B). Annexin V FITC/PI staining showed that the apoptosis rate of HCC cells treated with HPG-PCL-PTX monotherapy increased only slightly. However, when combined with BEZ235, the apoptosis rate was signifcantly enhanced(Fig. 7 D). Molecular investigations indicated that the expression level of pro-apoptotic proteins (caspase-9, caspase-3, caspase-7, Bad, Bax, Bim, Cyt C and Apaf-1) was increase and anti-apoptosis(Bcl-2) was decreased after HPG-PCL-PTX plus BEZ235 treated to HCC cells(Fig. 7 E). These results proved that HPG-PCL-PTX plus BEZ235 can promote the apoptosis of HCC cells and restore most of the sensitivity of these cells to PTX. HPG-PCL-PTX combined with BEZ235 enhanced the anti-tumor effect in vivo by inhibiting PI3K/mTOR and MAPK pathway Anti-tumor effects of HPG-PCL-PTX combined with BEZ235 in vivo were evaluated also in a murine xenograft model using HepG2R cells. Tumor tissues of each group were isolated and collected after 30-day treatment of tumor-bearing mice. The tumor growth of animals treated HPG-PCL-PTX combined with BEZ235 group was significantly inhibited compared with that in the HPG-PCL-PTX, BEZ235 and control groups ( p < 0.01) (Fig. 8 A). The molecular mechanism of the anti-tumor activity of HPG-PCL-PTX combined with BEZ235 was further examined by WB analysis of protein lysates from HepG2R xenografts. The results showed that HPG-PCL-PTX combined with BEZ235 group significantly reduced the phosphorylation levels of PI3K, Akt, S6K and Erk compared with levels in the control, paclitaxel and BEZ235 groups ( p < 0.01) (Fig. 8 B). There was no significant change in the body weight of the mice during the entire treatment period.(Fig. 8 C). Thus, we concluded that BEZ235 significantly enhanced the antitumor effect of paclitaxel in HCC in vivo , at least in part by inhibiting the PI3K/Akt/mTOR pathway. Discussion In this work, we developed the HPG-PCL deliver PTX, than combined with BEZ235 for activity against the HepG2 and HepG2R cells. At the cytological level, BEZ235 and HPG-PCL-PTX in combination had significantly more effect on proliferation and apoptosis than did either drug alone. Similarly, in vivo, HPG-PCL-PTX combined with BEZ235 treatment had a greater inhibitory effect on tumor growth than did either drug alone. Despite its potent and extensive antitumor activity, the efficacy of PTX is limited by resistance [ 29 ] . Thus, new strategies are needed to supress the progress of HCC. The mechanism of HCC resistance to treatment is complicated: the PI3K/Akt/mTOR pathway is activated in 30–50% of HCC [ 30 ] , and the MAPK pathway is also activated in HCC [ 31 ] . Activated PI3K/Akt/mTOR and MAPK induces cell survival and proliferation and induces HCC resistance [ 32 – 33 ] . Our study also confirmed that PI3K/Akt/mTOR and MAPK pathways in HCC cells were significantly activated after PTX resistance, promoting cell proliferation and inhibiting apoptosis. Presumably, HCC cells acquired resistance by promoting cell cycle progression and inhibiting apoptosis. CyclinB1 is a crucial regulator in G2/M transition, whose upregulation has been identified to confer resistance to PTX [ 34 – 35 ] . Consistently, increased cyclinB1 was observed in our established PTX-resistant cells. To further evaluate functional status of G2/M regulate factors, we also compared their expression level of parental cells and their resistant cells. The cyclinB1 accumulation and CDK1 de-phosphorylation mediating G2/M block [ 36 – 37 ] . According to our results, PTX-resistant cells had reduced responses to PTX, giving a hint that the acquisition of PTX resistance attenuated PTX-mediated inability of mitotic spindle formation. Furthermore, apoptosis triggered by mitotic arrest is one a major route for PTX-induced antitumor effects [ 38 ] . PTX activates caspase-dependent apoptotic pathways after inducing G2/M arrest in prostate cancer cells [ 39 ] . Given that mitosis stalling can initiate the apoptotic pathways, we tested whether apoptotic resistance occurred in HCC cells. Our research confirms that HepG2R cells had greater anti-apoptotic capacity, as indicated by reduced caspase-9, caspase-3 ,caspase7, Bim and Bax than in HepG2 cells [ 40 – 41 ] . Apoptotic induction has been considered as a candidate to treat PTX-resistant cancers [ 42 ] . Nevertheless, as we found that impact of PTX on these pro-apoptotic proteins was less effective in resistant cells than in parental, targeting PI3K/Akt/mTOR might be a more efficient option against PTX-resistant cancer than apoptotic induction. The free PTX in vitro did not decrease the phosphorylation levels of p-Akt, c-Raf, MEK and ERK kinases in HepG2 or HepG2R cells, which may be due to a stress response or self-protection mechanism. In other words, PTX stimulated the activation of the PI3K/Akt/mTOR pathway in HepG2 and HepG2R cells. These results confirmed previous findings that the classical pathway in HCC cells may crosstalk to stimulate activation of other signaling pathways [ 43 – 44 ] . In fact, the stimulation of Hepatocyte growth factor secretion in HCC cells by PTX promoted the phosphorylation of S6K and 4EBP1, which further stimulated the activation of the PI3K/Akt/mTOR pathway, thus supporting the hypothesis that the free PTX treatment may not effective against HCC. Therefore we prepared HPG-PCL nanoparticles loaded with PTX (HPG-PCL-PTX). When the polymer complexes passed through the circulatory system, PTX were delivered to HCC tissues through the high permeability and retention effect of the nanoparticles. Animal experiments show that treatment with HPG-PCL-PTX combined with BEZ235 significantly inhibited tumor growth; average tumor volume was significantly smaller than other treatment groups. Meanwhile, the body weight of the mice treated with HPG-PCL-PTX combination of BEZ235 group was not significantly decreased; this finding suggests that HPG-PCL-PTX combined with BEZ235 have advantages in drug delivery less delivery of the drug to non-targeted tissues, with attendant less toxicity and fewer side effects. Correspondingly, HPG-PCL-PTX combined with BEZ235 were enriched in HepG2R cells, this result would be expected to improve the anti-tumor effectiveness of the drugs. In fact, increasing research has found that Ras/Raf/MAPK and PI3K/Akt/mTOR are the main signaling pathways activated in HCC cells [ 45 ] . HPG-PCL-PTX combined with BEZ235 can reduce proliferation and induce apoptosis in HCC cell lines; in xenograft mouse models enhanced tumor necrosis [ 46 ] . Our results corroborate these findings that during the treatment of HCC, PTX alone inhibits the Ras/Raf/MAPK signaling pathway but has limited efficacy and is likely to cause desensitization of HCC to PTX. HPG-PCL-PTX combined with BEZ235 will increase the HCC cells inhibition effect of PTX, especially for PTX-resistant HCC. In summary, our results document that HPG-PCL-PTX combined with BEZ235 have significant anti-HCC effects in vitro and in vivo, especially in PTX-resistant HCC. Thus, BEZ235 combined with PTX of transportation via nanocarriers is a promising treatment for controlling HCC, especially for PTX-resistant HCC. Materials And Methods Reagents and antibodies PTX, 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-benzimidazolyl carbocyanine iodide (JC-1), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), Annexin V-FITC/PI Kit and DAPI were purchased from Sigma-Aldrich (St. Louis, MO, USA). BEZ235(Dactolisib) was obtained from MedChem Express (Monmouth Junction, NJ, USA). RPMI-1640 was obtained from Hyclone (Salt Lake City, UT, USA). FBS was purchased from Hangzhou Sijiqing Company. PI3K Antibody Kit (9655#), Akt Antibody Kit (9916#), mTOR Antibody Kit (9964#), Bcl-2 Family Antibody Kit (9942#), Apoptosis Antibody Kit (9915#), secondary goat anti-rabbit and anti-mouse antibodies were purchased from Cell Signal Technology (Danvers, MA, USA). Cyclin B1 and Cyclin-dependent-kinase (CDK1) were purchased from Abcam Biological Technology (USA). β-actin was obtained from Cell Signal Technology (Danvers, MA, USA). Cell lines and cell culture Human HepG2 cell line was bought from the American Type Culture Collection (Manassas, VA, USA). HepG2R, a cell line resistant to paclitaxel by HepG2 cells. The process of inducd HepG2R was as follows: When the cells were in the logarithmic growth phase, added PTX with a lower concentration for 24h, then performed cell inheritance, and repeatedly stimulated with this concentration until it was stable. Increasing the concentration of PTX continued to stimulate to a stable state. When the resistant strain treated with PTX reached 4–5 times the IC50 of the sensitive strain, the resistant strain was obtained. Synthesis of HPG-PCL The amphiphilic HPG derivative PCL-HPG was synthesized by anionic ring-opening polymerization as follows: First 1.5g (0.10 mmol) of dry PCL was weighed into a 50mL Schlenk flask and repeatedly evacuated several times with nitrogen gas using an anhydrous oxygen-free. Then 0.8mL of 25% potassium methanol solution was added and the temperature was gradually increased to 95°C while stirring. 10min later, the excess methanol was removed by re-evacuation. Subsequently, 4.5g (60.7mmol) of epichlorohydrin was added dropwise to the reaction system using a micro syringe pump under the protection of nitrogen. After the dropwise addition, the reaction was continued for 5h. At the end of the reaction, the product was dissolved with an appropriate amount of methanol and the acidified cation exchange resin was added to remove the potassium ions. The crude product was removed by rotary evaporation and lyophilised using a dialysis bag with a cut-off molecular weight of 3000 to give a clear yellow viscous solution. Preparation of HPG-PCL-PTX nanoparticles Weigh 500mg of HPG-PCL in 4mL of water, 10mg of PTX in 2mL of acetone. The PTX solution was added to the HPG-PCL solution drop by drop (as the PTX solution was added, the system gradually became cloudy, and the solution was clarified by adding acetone) and stirred at room temperature overnight. The drug-loaded nanoparticles were lyophilised by dialysis. characterization of HPG-PCL-PTX The average particle-diameter distribution and zeta potential of the nanospheres were tested by a Malvern Zetasizer Nano ZS (Malvern, Worcester, UK). The morphology of the NPs was observed with a transmission electron microscope (TEM, Tecnai F30, FEI Company, Hillsboro, OR, USA). The structure of the synthesized PLGA-PEG in CDCl3 was confirmed by the 1H NMR spectra (Varian Unity Inova 400 Mhz, Agilent Technologies, Inc., Santa Clara, CA, USA). To analysis drug loading content (LC%) of NPs, high-performance liquid chromatography (HPLC, LC 1200, Agilent Technologies, Santa Clara, CA, USA) was applied. Calculate LC% by the following equation : LC%=(BEZ235weight inNPs)/(totalNPsweight)×100% Molecular targeted The HPG-PCL-PTX was labeled with FITC, then co-cultured with HepG2 and HepG2R cells. The nanomedicine with unmodified targeting molecules used as a control, observe the endocytosis effect of the two cell lines on nano-drugs at 0.5h, 1h, 2h and 4h, respectively. Cell viability HepG2 and HepG2R cells (5000–10000 per well) were plated into 96-well plates and incubated overnight in a 5% CO2 incubator at 37°C. After indicated drugs exposured for 24 h, cell viability was measured using MTT assay according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a spectrophotometer. IC50 was calculated by Graphpad Prism Version 5.0 software. The resistant index (RI) was calculated utilizing the following formula: RI = IC50 of resistant cells/IC50 of parental cells. Colony-formation assay Cells were cultured into 6-well plates at a density of 1000 cells per well to adhere overnight, then the cells were treated with the drugs for 24h. After about two weeks, the cell colonies were clearly visible. The medium was aspirated, colonies were fixed in 4% paraformaldehyde for 15min and stained with 0.1% crystal violet for 30min. Cell colonies were counted, and the number cultured with drugs was compared with the control. Cell cycle analysis Propidium iodide (PI) was used to stain the DNA content. After drug interventions for 24 h, cells were collected, washed with PBS, and fixed with 70% precooled ethanol. Before testing, cells were washed with PBS three times, added 50µg/ml PI and 100µg/ml RNase A in the dark for 30min. Dye was removed, the cells were resuspended with PBS, followed by flow cytometry analysis (BD FACSCalibur, USA). The distribution of cells at specifific cell cycle stages was assessed with ModFit Version 3.0 software (Verity Software House, Topsham, ME). Apoptosis detection Cells were seeded in 24-well plates and incubated for overnight. After drug treatment for 24h, cells were collected after digestion and centrifugation, double-stained with Annexin V-FITC(10µL) and PI(5µL) at room temperature for 20min in the dark. Cell apoptosis was detected by flow cytometry within 1 h (BD Biosciences) and analyzed apoptotic rates using the Flow Jo software. Mitochondrial membrane potential (ΔΨm) measurement JC-1 is a sensitive probe for measuring mitochondrial membrane potential. The high/low mitochondrial membrane potential of cells treated with dugs determined red/green fuorescence intensity excited by polymer/monomer of JC-1, from which the mitochondrial apoptosis level of the cells was analyzed. Cells were cultured in 24-well plates overnight, treated with drugs for 24h, counterstained with DAPI and JC-1, examined with fluorescence microscope. Besides, cells treated with drug were stained with 10µg/ml JC-1 for 30min at room temperature and analyzed with flow cytometry for changes in ΔΨm. Western blot Total protein was extracted from cells lysed with radioim munoprecipitation bufer (RIPA, Beyotime Biotechnology, Shanghai, China) containing a protease inhibitor cocktail (Beyotime Biotechnology, Shanghai, China) and centrifuged. The BCA protein assay kit (Biosharp, Hefei, China) was used for measuring protein concentration. Soluble lysates containing about 20 µg proteins per sample were resolved with sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (Millipore, Sigma, USA). After blocking using 5% skim milk, membranes were probed with primary antibodies (dilutions were 1:1000) at 4°C overnight and secondary antibodies (1:2000) at room temperature for 1 h. Protein bands were visualized with an ECL luminescent detection kit (Thermo Fisher Scientific Waltham, MA, USA) and images were captured with a gel imager Bio-Rad (Hercules, CA, USA). Quantified with Image J Version 1.48 software (NIH, Bethesda, MD). The protein content of β-actin was used as a loading control. Xenograft studies All animal experiments were approved by the Animal Experimental Ethics Committee of Anhui University of Science and Technology and were carried out in accordance with appropriate procedures. Animal experiments were carried out in the SPF-level animal room of the Central Laboratory of Medical School Anhui University of Science and Technology (NO: AUST201810088). 100µl of HepG2 cell suspension (2×10 7 /ml) were injected subcutaneously into the dorsal right side of 5-week-old female BALB/c nude mice (Vital River Laboratories, Beijing, China). When tumor volume reached 100 mm3, mice were randomized into five groups: control group: 100µl saline daily, ip; HPG-PCL-PTX group: 5mg/kg weekly, ip; BEZ235 group: 45mg/kg daily, oral treatment. (The dose of BEZ235 used in vivo was based on the specifications of Selleck Chemicals); HPG-PCL-PTX + BEZ235 group: PTX (5mg/kg weekly, ip) plus BEZ235 (45mg/kg daily, oral treatment). Each group of rats received treatment for 28 days, and tumor size and body weight were measured every 3 days. Tumor volume was calculated according to the formula: V = L×W 2 ×1/2 (V, volume; L, length of tumor; W, width of tumor). Mice were sacrificed after the treatment; tumor tissues were isolated; protein was extracted for WB detection. Statistical analysis All analyses were performed with SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). All experiments were repeated three times independently and analyzed by Student t-test or two-way ANOVA using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). Data are expressed as mean ± SD. p < 0.05 was taken as statistically significant. References Alqahtani A, Khan Z, Alloghbi A, Said Ahmed TS, Ashraf M & ammouda DM. Hepatocellular Carcinoma: Molecular Mechanisms and Targeted Therapies. Medicina (Kaunas) 55 , 526(2019). Yang JD, Hainaut P, Gores GJ, Amadou A, Plymoth A & Roberts LR. A global view of hepatocellular carcinoma: trends, risk, prevention and management. Nat Rev Gastroenterol Hepatol 16 , 589–604(2019). Zou H, Li L, Garcia Carcedo I, Xu ZP, Monteiro M & Gu W. Synergistic inhibition of colon cancer cell growth with nanoemulsion-loaded paclitaxel and PI3K/mTOR dual inhibitor BEZ235 through apoptosis. Int J Nanomedicine 11 , 1947–58(2016). Chakraborty S, Dlie ZY, Chakraborty S, Roy S, Mukherjee B, Besra SE. et al. Aptamer-Functionalized Drug Nanocarrier Improves Hepatocellular Carcinoma toward Normal by Targeting Neoplastic Hepatocytes. Mol Ther Nucleic Acids 20 , 34–49(2020). Sanchez-Carranza JN, González-Maya L, Razo-Hernández RS, Salas-Vidal E, Nolasco-Quintana NY, Clemente-Soto AF. et al. Achillin Increases Chemosensitivity to Paclitaxel, Overcoming Resistance and Enhancing Apoptosis in Human Hepatocellular Carcinoma Cell Line Resistant to Paclitaxel (Hep3B/PTX). Pharmaceutics 11 (10), 512(2019). Saville MW, Lietzau J, Pluda JM, Feuerstein I, Odom J, Wilson WH. et al. Treatment of HIV-associated Kaposi's sarcoma with paclitaxel. Lancet 346 (8966), 26 – 8(1995). Gupta N, Hatoum H & Dy GK. First line treatment of advanced non-small-cell lung cancer - specific focus on albumin bound paclitaxel. Int J Nanomedicine 9 , 209 – 21(2014). Gan H, Chen L, Sui X, Wu B, Zou S, Li A. et al. Enhanced delivery of sorafenib with anti-GPC3 antibody-conjugated TPGS-b-PCL/Pluronic P123 polymeric nanoparticles for targeted therapy of hepatocellular carcinoma. Mater Sci Eng C Mater Biol Appl 91 , 395–403(2018). Wu B, Liang Y, Tan Y, Xie C, Shen J, Zhang M. et al. Genistein-loaded nanoparticles of star-shaped diblock copolymer mannitol-core PLGA-TPGS for the treatment of liver cancer. Mater Sci Eng C Mater Biol Appl 59 , 792–800(2016). Lin Q, Yang Y, Hu Q, Guo Z, Liu T, Xu J. et al. Injectable supramolecular hydrogel formed from α-cyclodextrin and PEGylated arginine-functionalized poly(l-lysine) dendron for sustained MMP-9 shRNA plasmid delivery. Acta Biomater 49 , 456–471(2017). Mei L, Zhang Y, Zheng Y, Tian G, Song C, Yang D. et al. A Novel Docetaxel-Loaded Poly (ε-Caprolactone)/Pluronic F68 Nanoparticle Overcoming Multidrug Resistance for Breast Cancer Treatment. Nanoscale Res Lett 4 (12), 1530–9(2009). Sharma S, Verma A, Pandey G, Mittapelly N & Mishra PR. Investigating the role of Pluronic-g-Cationic polyelectrolyte as functional stabilizer for nanocrystals: Impact on Paclitaxel oral bioavailability and tumor growth. Acta Biomater 26 , 169 – 83(2015). Awasthi N, Yen PL, Schwarz MA & Schwarz RE. The efficacy of a novel, dual PI3K/mTOR inhibitor NVP-BEZ235 to enhance chemotherapy and antiangiogenic response in pancreatic cancer. J Cell Biochem 113 (3), 784–91(2012). Byun DS, Cho K, Ryu BK, Lee MG, Park JI, Chae KS. Et al. Frequent monoallelic deletion of PTEN and its reciprocal associatioin with PIK3CA amplification in gastric carcinoma. Int J Cancer 104 (3):318–27(2003). Li VS, Wong CW, Chan TL, Chan AS, Zhao W, Chu KM. et al. Mutations of PIK3CA in gastric adenocarcinoma. BMC Cancer 5 , 29(2005). Elsadek B & Kratz F. Impact of albumin on drug delivery–new applications on the horizon. J Control Release 157 (1), 4–28(2012). Gradishar WJ, Tjulandin S, Davidson N, Shaw H, Desai N, Bhar P. et al. Phase III trial of nanoparticle albumin-bound paclitaxel compared with polyethylated castor oil-based paclitaxel in women with breast cancer. J Clin Oncol 23 (31), 7794–803(2005). Blanco E, Sangai T, Wu S, Hsiao A, Ruiz-Esparza GU, Gonzalez-Delgado CA. et al. Colocalized delivery of rapamycin and paclitaxel to tumors enhances synergistic targeting of the PI3K/Akt/mTOR pathway. Mol Ther 22 (7), 1310–1319(2014). Nazio F, Strappazzon F, Antonioli M, et al. mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6. Nat Cell Biol 15 , 406–416(2013). Wu YY, Wu HC, Wu JE, Huang KY, Yang SC, Chen SX. Et al. The dual PI3K/mTOR inhibitor BEZ235 restricts the growth of lung cancer tumors regardless of EGFR status, as a potent accompanist in combined therapeutic regimens. J Exp Clin Cancer Res 38 (1), 282(2019). Meng W, Wang B, Mao W, Wang J, Zhao Y, Li Q. et al. Enhanced efficacy of histone deacetylase inhibitor panobinostat combined with dual PI3K/mTOR inhibitor BEZ235 against glioblastoma. Nagoya J Med Sci 81 (1), 93–102(2019). Aung W, Tsuji AB, Sudo H, Sugyo A, Ukai Y, Kouda K. et al. Combined treatment of pancreatic cancer xenograft with 90Y-ITGA6B4-mediated radioimmunotherapy and PI3K/mTOR inhibitor. World J Gastroenterol 23 (42), 7551–7562(2017). Chen L, Jin T, Zhu K, Piao Y, Quan T, Quan C. et al. PI3K/mTOR dual inhibitor BEZ235 and histone deacetylase inhibitor Trichostatin A synergistically exert anti-tumor activity in breast cancer. Oncotarget 8 (7), 11937–11949(2017). Zhu Y, Tian T, Zou J, Wang Q, Li Z, Li Y. et al. Dual PI3K/mTOR inhibitor BEZ235 exerts extensive antitumor activity in HER2-positive gastric cancer. BMC Cancer 15 , 894(2015). Wu B, Li A, Zhang Y, Liu X, Zhou S, Gan H. et al. Resistance of hepatocellular carcinoma to sorafenib can be overcome with co-delivery of PI3K/mTOR inhibitor BEZ235 and sorafenib in nanoparticles. Expert Opin Drug Deliv 17 (4), 573–587(2020). Chen D, Lin X, Zhang C, Liu Z, Chen Z, Li Z. et al. Dual PI3K/mTOR inhibitor BEZ235 as a promising therapeutic strategy against paclitaxel-resistant gastric cancer via targeting PI3K/Akt/mTOR pathway. Cell Death Dis 9 (2), 123(2018). Liu X, Xie C, Li A, Zhang Y, Liu X, Zhou S. et al. BEZ235 enhances chemosensitivity of paclitaxel in hepatocellular carcinoma through inhibiting the PI3K/Akt/mTOR pathway. Am J Transl Res 11 (12), 7255–7271(2019). Chang Z, Shi G, Jin J, Guo H, Guo X, Luo F. et al. Dual PI3K/mTOR inhibitor NVP-BEZ235-induced apoptosis of hepatocellular carcinoma cell lines is enhanced by inhibitors of autophagy. Int J Mol Med 31 (6), 1449–56(2013). Shen L, Shan YS, Hu HM, Price TJ, Sirohi B, Yeh KH. et al. Management of gastric cancer in Asia: resource-stratified guidelines. Lancet Oncol 14 (12), e535-47(2013). Cervello M, Emma MR, Augello G, Cusimano A, Giannitrapani L, Soresi M. et al. New landscapes and horizons in hepatocellular carcinoma therapy. Aging (Albany NY) 12 (3), 3053–3094(2020). Moon H & Ro SW. MAPK/ERK Signaling Pathway in Hepatocellular Carcinoma. Cancers (Basel) 13 (12), 3026(2021). Bidkhori G, Benfeitas R, Klevstig M, Zhang C, Nielsen J, Uhlen M. et al. Metabolic network-based stratification of hepatocellular carcinoma reveals three distinct tumor subtypes. Proc Natl Acad Sci U S A 115 (50), E11874-E11883(2018). Wani NA, Zhang B, Teng KY, Barajas JM, Motiwala T, Hu P. et al. Reprograming of Glucose Metabolism by Zerumbone Suppresses Hepatocarcinogenesis. Mol Cancer Res 16 (2), 256–268(2018). Ou Y, Ma L, Ma L, Huang Z, Zhou W, Zhao C. et al. Overexpression of cyclin B1 antagonizes chemotherapeutic-induced apoptosis through PTEN/Akt pathway in human esophageal squamous cell carcinoma cells. Cancer Biol Ther 14 (1), 45–55(2013). Yuan J, Krämer A, Matthess Y, Yan R, Spänkuch B, Gätje R. et al. Stable gene silencing of cyclin B1 in tumor cells increases susceptibility to taxol and leads to growth arrest in vivo. Oncogene 25 (12), 1753–62(2006). Chang WL, Yu CC, Chen CS & Guh JH. Tubulin-binding agents down-regulate matrix metalloproteinase-2 and – 9 in human hormone-refractory prostate cancer cells–a critical role of Cdk1 in mitotic entry. Biochem Pharmacol 94 (1), 12–21(2015). Giovinazzi S, Bellapu D, Morozov VM & Ishov AM. Targeting mitotic exit with hyperthermia or APC/C inhibition to increase paclitaxel efficacy. Cell Cycle 12 (16), 2598–607(2013). Harley ME, Allan LA, Sanderson HS & Clarke PR. Phosphorylation of Mcl-1 by CDK1-cyclin B1 initiates its Cdc20-dependent destruction during mitotic arrest. EMBO J 29 (14), 2407–20(2010). Wang C, Huang SB, Yang MC, Lin YT, Chu IH, Shen YN. et al. Combining paclitaxel with ABT-263 has a synergistic effect on paclitaxel resistant prostate cancer cells. PLoS One 10 (3), e0120913(2015). Duran GE, Wang YC, Moisan F, Francisco EB & Sikic BI. Decreased levels of baseline and drug-induced tubulin polymerisation are hallmarks of resistance to taxanes in ovarian cancer cells and are associated with epithelial-to-mesenchymal transition. Br J Cancer 116 (10), 1318–1328(2017). Bae T, Weon KY, Lee JW, Eum KH, Kim S & Choi JW. Restoration of paclitaxel resistance by CDK1 intervention in drug-resistant ovarian cancer. Carcinogenesis 36 (12), 1561–71(2015). Wang L, Li H, Ren Y, Zou S, Fang W, Jiang X. et al. Targeting HDAC with a novel inhibitor effectively reverses paclitaxel resistance in non-small cell lung cancer via multiple mechanisms. Cell Death Dis 7 (1), e2063(2016). Ebrahim HM, El-Rouby MN, Morsy ME, Said MM & Ezz MK. The Synergistic Cytotoxic Effect of Laser-Irradiated Gold Nanoparticles and Sorafenib Against the Growth of a Human Hepatocellular Carcinoma Cell Line. Asian Pac J Cancer Prev 20 (11), 3369–3376(2019). Toh TB, Lim JJ, Hooi L, Rashid MBMA & Chow EK. Targeting Jak/Stat pathway as a therapeutic strategy against SP/CD44 + tumorigenic cells in Akt/β-catenin-driven hepatocellular carcinoma. J Hepatol 72 (1), 104–118(2020). Gedaly R, Angulo P, Hundley J, Daily MF, Chen C, Koch A. et al. PI-103 and sorafenib inhibit hepatocellular carcinoma cell proliferation by blocking Ras/Raf/MAPK and PI3K/AKT/mTOR pathways. Anticancer Res 30 (12), 4951–8(2010). Rimassa L, Pressiani T & Merle P. Systemic Treatment Options in Hepatocellular Carcinoma. Liver Cancer 8 (6), 427–446(2019). Declarations Acknowledgements This work was supported by Wu Jieping Medical Foundation Clinical Research Special Fund(no. 320.6750.2022-02-2). Conflict of interest statement The authors declare that they have no conflict of interest. Author contribution statement Xiaolong Tang performed study concept and design; Xueke Liu performed development of methodology and writing, review and revision of the paper; Yingping Wu, Wei Xu, Lele Li provided acquisition, analysis and interpretation of data, and statistical analysis; Xiaoping Xia provided technical and material support. All authors read and approved the final paper. Ethics statement The animal experiments were approved by the Animal Experimental Ethics Committee of Anhui University of Science and Technology and were carried out in the SPF-level animal room of the Central Laboratory of Medical School Anhui University of Science and Technology (NO: AUST201810088). Data availability statement All data and materials are available without restriction. Supplementary Files Originalfulllengthwesternblots.zip Original full length western blots Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1793410","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":116246997,"identity":"a07052e4-ae47-40d1-86f4-2a09d46780ab","order_by":0,"name":"Xueke Liu","email":"","orcid":"","institution":"Fourth Affiliated Hospital, College of Medicine, Zhejiang University, Jinhua, 322000, China","correspondingAuthor":false,"prefix":"","firstName":"Xueke","middleName":"","lastName":"Liu","suffix":""},{"id":116246998,"identity":"97836dec-ffc7-4c6b-b6fe-8194f38b2e01","order_by":1,"name":"Yingping wu","email":"","orcid":"","institution":"Fourth Affiliated Hospital, College of Medicine, Zhejiang University, Jinhua, 322000, China","correspondingAuthor":false,"prefix":"","firstName":"Yingping","middleName":"","lastName":"wu","suffix":""},{"id":116246999,"identity":"b4d1839f-f1a6-4a53-b49b-fe9beddc5b24","order_by":2,"name":"We Xu","email":"","orcid":"","institution":"Fourth Affiliated Hospital, College of Medicine, Zhejiang University, Jinhua, 322000, China","correspondingAuthor":false,"prefix":"","firstName":"We","middleName":"","lastName":"Xu","suffix":""},{"id":116247000,"identity":"07c97d9b-d70c-4aac-98b8-02e0c50e878a","order_by":3,"name":"Lele Li","email":"","orcid":"","institution":"Fourth Affiliated Hospital, College of Medicine, Zhejiang University, Jinhua, 322000, China","correspondingAuthor":false,"prefix":"","firstName":"Lele","middleName":"","lastName":"Li","suffix":""},{"id":116247001,"identity":"508f6e4f-1121-4385-a322-9e982b643c27","order_by":4,"name":"Xiaoping Xia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYBACg8NgEsxgfMDAA+IlEK+F2YAoLZINCAabBIRJQAs/O/PDxwUFd+z62ZmfVf6QOQwUyTFg+LkDtxY2ZjZj4xkGz5LbmNnMbkjwHGaQ7HljwNh7Bp8WBjNpHoPDySDGDQOgFoMbOQbMjG14HMbM/g2sBcQoSABqsSekRbKZB2yLHYjBcABkiwQBLQaHeYqBfjmcAGJINvCk80iceVZwsBeflvPHNz4u+HPYHsT4+LPHWo6/PXnjg594tIAAMxAnNoBYjD2QyDyAXwNEiz2E+YOQ2lEwCkbBKBiJAAB9d0o1w9jrJAAAAABJRU5ErkJggg==","orcid":"","institution":"Fourth Affiliated Hospital, College of Medicine, Zhejiang University, Jinhua, 322000, China","correspondingAuthor":true,"prefix":"","firstName":"Xiaoping","middleName":"","lastName":"Xia","suffix":""},{"id":116247002,"identity":"ce81b96c-06c4-44f4-ad72-70ba91e0dd65","order_by":5,"name":"Xiaolong Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIie3RsYrCQBCA4QkLCwertiMR8wqRQBC080VWhLVR0M5ORUkKY3/3FpbXXWRgbXLYWkZ8Ae1Smv6OJHYW+9Xzw+4MgGG8Ie7Q6X5f9NsNoGMqs355UkeurM9Eec21HrnXSJUnbRQ++9jS8ECJ30w5VXiYHaibtSJrpWN/IQVBI9zJ4qRFJ2/+PWZWFKuL7I4Bk99DcQJK2l9JjzOM9UWKHrg4LUsmrl0LmODONZhJziokOPHzZIACiIPkgwqJ0KNOvmQXQTMcRkqU/sUJN8c0P+XyJz4/HlmW3zTcFyd/iNfGDcMwjH89Ad/bTWTIwYliAAAAAElFTkSuQmCC","orcid":"","institution":"Clinical Laboratory Medicine, First Affiliated Hospital, Anhui University of Science \u0026 Technology, Huainan 232001, China.","correspondingAuthor":true,"prefix":"","firstName":"Xiaolong","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2022-06-25 02:19:39","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true,"coiExplicitlySet":false},"doi":"10.21203/rs.3.rs-1793410/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1793410/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":23231544,"identity":"68f76e6b-e219-4c77-b879-66b24368a786","added_by":"auto","created_at":"2022-06-29 14:39:12","extension":"tif","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8621868,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation process and physical characterization of NPs. (A) 1H NMR spectra of HPG-PCL copolymer. (B) Size distribution of the HPG-PCL and HPG-PCL-PTX detected by DLS. (C) Transmission electron micrographs (TEM) of NHPG-PCL and HPG-PCL-PTX. (D) HPLC profile of HPG-PCL-PTX. Bar: 100 nm.\u003c/p\u003e","description":"","filename":"Figure101.tif","url":"https://assets-eu.researchsquare.com/files/rs-1793410/v1/b2f1c2840b85ad1cafc91b0f.tif"},{"id":23231551,"identity":"24236cd7-0313-4b10-bbe6-5c66dcd3c493","added_by":"auto","created_at":"2022-06-29 14:39:12","extension":"tif","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8935817,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of cell proliferation viability. (A) a Cell viability of HepG2 and HepG2R cells after exposure to PTX for 24 h. (B and C) Colony formation of HepG2 cell in different treatment. Data expressed as mean±SD, n=3 (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure201.tif","url":"https://assets-eu.researchsquare.com/files/rs-1793410/v1/3a866e0ad9a60e3491e7ebb9.tif"},{"id":23231545,"identity":"96918f0b-2dfd-44c7-813c-14ef6470524f","added_by":"auto","created_at":"2022-06-29 14:39:12","extension":"tif","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5722445,"visible":true,"origin":"","legend":"\u003cp\u003eThe uptake efficiency of nano drug (HPG-PCL-PTX) by cells. Confocal microscopy was used to detect the content of tracer HPG-PCL-PTX-FITC in the HepG2 and HepG2R cell lines, respectively. blue, 4′,6-diamidino-2-phenylindole (DAPI). Data expressed as mean±SD, n=3 (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure301.tif","url":"https://assets-eu.researchsquare.com/files/rs-1793410/v1/d0aa049083fb94fd2d8d1043.tif"},{"id":23232727,"identity":"fe163f59-fb85-43e7-86da-74c7dd08f118","added_by":"auto","created_at":"2022-06-29 14:49:12","extension":"tif","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10255373,"visible":true,"origin":"","legend":"\u003cp\u003eHepG2 and HepG2R cells were incubated with PTX at different time individualy, cell lysates were collected, western blot detected the phosphorylation levels of key molecules in HepG2 and HepG2R cells. (A) PI3K/Akt and Ras/Raf/MAPK pathway; (B) mTOR and autophagy pathway. (C) Signaling pathway regulation pattern chart.\u003c/p\u003e","description":"","filename":"Figure401.tif","url":"https://assets-eu.researchsquare.com/files/rs-1793410/v1/f9ed025cb85a7de832d0b6d4.tif"},{"id":23232017,"identity":"3b3d6b26-f13a-42a9-a850-50ad0a88a56c","added_by":"auto","created_at":"2022-06-29 14:44:12","extension":"tif","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9134686,"visible":true,"origin":"","legend":"\u003cp\u003eHepG2 and HepG2R cells were incubated under various treatment group for 24 h, cell lysates were collected, western blot detected the phosphorylation levels of key molecules in HepG2 and HepG2R cells. (A) PI3K/Akt and Ras/Raf/MAPK pathway; (B) mTOR and autophagy pathway. (C) The role of BEZ235 on the molecular regulation pattern chart of signaling pathways.\u003c/p\u003e","description":"","filename":"Figure501.tif","url":"https://assets-eu.researchsquare.com/files/rs-1793410/v1/e1b8dbd6781439c3776b2067.tif"},{"id":23231548,"identity":"b20eaa5e-02b2-49a9-83ac-ee0d82bac7cf","added_by":"auto","created_at":"2022-06-29 14:39:12","extension":"tif","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4731941,"visible":true,"origin":"","legend":"\u003cp\u003eCell-cycle analysis of HepG2 and HepG2R. (A and B) After HepG2 and HepG2R cells were exposed to each treatment group for 24h, propidium iodide stained, Cell cycle distribution was analyzed by flow cytometry. (C and D) Analysis of Cyclin Expression Levels by Western Blotting. Data expressed as mean±SD, n=3 (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure601.tif","url":"https://assets-eu.researchsquare.com/files/rs-1793410/v1/92d76baf56a52b86b44a8e4b.tif"},{"id":23232019,"identity":"e3752cb4-ccaf-4a69-9781-2d71b22ef066","added_by":"auto","created_at":"2022-06-29 14:44:12","extension":"tif","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":14634258,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of HPG-PCL-PTX combined with BEZ235 on apoptosis of HepG2 cells. (A) The ΔΨm of HCC cells were dectected by flow cytometry; (B) The apoptosis of HCC celss were dectected by flow cytometry; (C) HCC cells were counterstained with JC-1 and DAPI and observed with fluorescence microscopy; (D) HCC cells were counterstained with DAPI/Annexin V-FITC/PI and observed with fluorescence microscopy; (E) The expression levels of apoptotic proteins (Bad, Bax, Bak, Bim, Bid, Bik, puma) and caspase proteins (caspase-9, -3, -7) in each treatment group were analyzed by WB. (F) Apoptotic molecule regulation pattern chart. Data expressed as mean±SD, n=3 (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure701.tif","url":"https://assets-eu.researchsquare.com/files/rs-1793410/v1/cce1e6a7b2304ad2b6173471.tif"},{"id":23231546,"identity":"995a4ea4-7364-4a6f-94c4-930b8338731e","added_by":"auto","created_at":"2022-06-29 14:39:12","extension":"tif","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1356176,"visible":true,"origin":"","legend":"\u003cp\u003eHPG-PCL-PTX plus BEZ235 significantly enhanced the anti-tumor effect in vivo by PI3K/mTOR double inhibition. (A) tumor tissues of each group were isolated and collected after 30-day-treatment of tumor-bearing mice. (B) Partial tumor tissue was lysed to extract tissue protein for WB analysis. (C) Body weight change in mice treated for 30 days. Data expressed as mean±SD, n=4 (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure801.tif","url":"https://assets-eu.researchsquare.com/files/rs-1793410/v1/14fb813e6b1faa0c67df026d.tif"},{"id":23232744,"identity":"e433d0fe-1c44-4903-8e60-92ef72099156","added_by":"auto","created_at":"2022-06-29 14:49:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":949692,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1793410/v1/2b7e2f18-ac93-49e7-bfba-a8f68cb8506f.pdf"},{"id":23232021,"identity":"c3089d25-dbe6-44b4-8778-b4fb64f2fb70","added_by":"auto","created_at":"2022-06-29 14:44:12","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":49590663,"visible":true,"origin":"","legend":"\u003cp\u003eOriginal full length western blots\u003c/p\u003e","description":"","filename":"Originalfulllengthwesternblots.zip","url":"https://assets-eu.researchsquare.com/files/rs-1793410/v1/68aa9459ce134412a5df4d2e.zip"}],"financialInterests":"","formattedTitle":"\u003cp\u003eBEZ235 enhances antitumor efficacy of HPG-PCL-PTX in HCC by suppressed the PI3K/Akt/mTOR and MAPK pathway\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumors worldwide\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Like many other cancers, acquired resistance is the biggest hurdles for effective treatments\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Molecular targeted therapy for the signaling pathways that induce to resistance of HCC is a novel and promising treatment approach. Identification of the key target molecules and signaling pathways responsible is crucial in order to develop effective targeted therapies against HCC\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePaclitaxel (PTX) is an approved chemotherapeutic in the UK for breast, lung, prostate and other types of solid tumors\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, but the low solubility hinders its application in the clinic. In addition, PTX cause some side effects such as hypersensitivity reactions, myelosuppression, and peripheral neuropathy\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. This makes the nano-delivery system a useful tool to assist with PTX solubility and delivery.\u003c/p\u003e \u003cp\u003eHyperbranched polyglycidyl ethers (HPG) have a wide range of applications in the biomedical field due to their low viscosity, long in vivo circulation time and large number of terminal modifiable groups\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. In this study, we first synthesize an amphiphilic polymer PCL-HPG using a degradable multi-armed polycaprolactone (PCL) as the core and a bisulfide bonded HPG as the arm, which uses a hydrophobic core PCL to load hydrophobic drugs, allowing the drug-loaded micelles to be stable in vivo and to achieve long circulation times, and increases the sensitivity of tumour cells to chemotherapeutic drugs to a certain extent\u003csup\u003e[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) signaling pathways play a central role for many tumor types in tumor cell proliferation, motility, invasion, metabolism and survival\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Approximately 10% of HCC patients carry a PIK3CA mutation in HCC\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, these mutations can activate PI3K and can lead to the activation of Akt via phosphorylation at Thr308 through PDK1 or/and at Ser473 through the mTOR associated with Rictor (the mTORC2 complex)\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Activated Akt regulates key downstream effectors including mTOR associated with Raptor (mTORC1 complex), p70S6 kinase and 4E-BP1\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Rapamycin and its analogous inhibit mTORC1 signaling and activate Akt signaling via mTORC2 related negative feedback loop. Abnormal activation of Akt and mTOR can reduce the effectiveness of anti-tumor drugs through promoting cellular proliferation, cell-cycle progression, inhibits the expression of mitochondrial pro-apoptotic-related proteins and prevents initiation of the pro-apoptotic caspase pathway, thus promoting cell survival\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInterestingly, mTOR kinase is an important regulator of inducing autophagy. Activated mTOR (Akt and MAPK signaling) inhibits autophagy, while negative regulation of mTOR (AMPK and p53 signaling) promotes autophagy. mTORC1 inhibits the initiation of autophagy through the ULK complex. mTORC1 induces phosphorylation of ULK1 at Ser758, represses the interaction between ULK1 and AMPK, and inhibits the activity of the ULK1 complex by phosphorylating ATG13, inducing phosphorylation of AMBRA1 to inhibit autophagy\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Thus, the combined inhibition of both PI3K and mTOR might be necessary for effective treatment of cancer.\u003c/p\u003e \u003cp\u003eNVP-BEZ235 (BEZ235), a novel dual PI3K/mTOR inhibitor, inhibits the catalytic subunit p110a of PI3K by competing at its ATP binding site and other class 1 PI3K enzymes and also inhibits the catalytic activity of mTOR\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. It was recently reported to mediate some anti-tumor effects in experimental pancreatic cancer\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, breast cancer\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e and gastric cancer\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTaken together, targeting PI3K/mTOR or MAPK signaling is a promising option. We evaluated targeting the PI3K/mTOR or MAPK pathways antitumor efficacy of BEZ235 combined with HPG-PCL-PTX in vivo and in vitro, determine more effective HCC therapeutic strategy. Our work provides ideas and directions for HCC resistance further research.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of HPG-PCL-PTX\u003c/h2\u003e \u003cp\u003eTo improve the ratio of drug concentration and effective combination in target cells and to overcome the short plasma half-life and adverse side effects of drugs, we applied HPG-PCL polymer nanoparticles to load PTX (PTX 5.0\u0026micro;M) to deliver PTX to HCC. From the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, it can be seen that the chemical shifts of 1.3, 1.6, 2.3 and 3.99ppm are the methylene proton absorption peaks at different positions on 4-arms-PCL, respectively. The proton absorption peak at the chemical shift of 33.4ppm is attributed to the methylene and methine groups of HPG, and the absorption peak at 4.6ppm is attributed to the hydroxyl group of HPG. The successful synthesis of HPG-PCL was demonstrated. By analyzing the particle size of the nanoparticles with DLS, it could be seen that the particle size of drug-loaded nanoparticles (HPG-PCL-PTX) was approximately 80\u0026thinsp;~\u0026thinsp;100nm diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Transmission electron microscopic images (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) reveal that the HPG-PCL and HPG-PCL-PTX are mostly spherical, relatively uniform in size, and have good monodispersity. Through the peak area in HPLC figure to calculate the drug loading rate is 2.2% and 8.2%(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBEZ235 combined with HPG-PCL-PTX inhibits proliferation of HCC cells\u003c/h2\u003e \u003cp\u003eChemotherapy normally causes severe side effects, even in combination treatments with different drugs. Nanodelivery of drugs may provide a solution to reduce these side effects by further decreasing the dosage. To achieve this, we used HPG-PCL to load PTX combined with BEZ235 to further increase their efficacy. PTX-resistant cell sublines HepG2R were established using exposure to escalating doses of PTX. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA shows half-maximal inhibitory concentration (IC50) values. In addition, clone formation experiments showed that BEZ235 combined with HPG-PCL-PTX can efectively inhibit the proliferation of HepG2 and HepG2R cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These data confirmed that HPG-PCL-PTX combined with BEZ235 has a synergistic treatment effect on HCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTargeting of HPG-PCL-FITC on HCC cells\u003c/h2\u003e \u003cp\u003eTo evaluate the uptake efficiency of nano drug (HPG-PCL-PTX) by cells, confocal microscopy was used to detect the content of tracer nanoparticle (HPG-PCL-PTX-FITC) in the HepG2 and HepG2R cell lines at defined time points, respectively. Timed visualization detected differences in cellular uptake by HPG-PCL-PTX. After incubation for 0.5 h, 1h, 2h, 4h, the endocytosis rates of HepG2 to HPG-PCL-PTX were 15.37%, 38.42%, 57.16%, 89.78%; the endocytosis rates of HepG2R to HPG-PCL-PTX were 9.64%, 17.92%, 48.57%, 85.34%. With the prolongation of time, the phagocytosis rate of HPG-PCL-PTX by hepG2 and HepG2R cells tended to be the same, both peaked at 4h(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results indicated that the efficient endocytosis of HPG-PCL-PTX by the cells resulted in higher cellular internalization of the drug.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePTX induces abnormal activation of PI3K/mTOR and MAPK pathways in HepG2 and HepG2R cells\u003c/h2\u003e \u003cp\u003ePrevious studies have noted that the involvement of the PI3K/mTOR and MAPK pathways in PTX resistance, and both pathways are further activated in PTX-resistant HCC cells compared with parental cells\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. To determine whether the resistance to PTX of HCC cells is related to activation of PI3K/mTOR and MAPK pathway, we detected the phosphorylation levels of key molecules on the pathways in HepG2 and HepG2R cells by Western blot. The results showed that the treatment of PTX on HepG2 cells caused stress activation of key molecules in PI3K/mTOR and MAPK pathways, and peaked at 24h. While, the effect of PTX on HepG2R cells led to the stable activation of key molecules in this pathway after 24h(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Thus, PTX resistance in HCC may be due to stable activation of PI3K/mTOR and MAPK signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBEZ235 combination of HPG-PCL-PTX significantly inhibits activation of PI3K/mTOR and MAPK pathway in HCC cells\u003c/h2\u003e \u003cp\u003eTo understand the therapeutic potential of targeting these two pathways to treat HCC cells, we assessed the PI3K/mTOR/Erk phosphorylation levels after 24h of PI3K/mTOR dual inhibitor BEZ235 in HCC cells by Western blot. The results showed that the BEZ235 combined with HPG-PCL-PTX effectively inhibted the PI3K/mTOR phosphorylation in HCC cells(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). However, when BEZ235 combined with PTX or HPG-PCL-PTX, these drugs elicited higher inhibition on Erk activity in resistant cells, yet in parental cells augmented Erk activity(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These might be due to that targeting PI3K/mTOR and MAPK pathways would induce re-activation of Akt/Erk in progenitor cells, whereas after the acquisition of PTX resistance, this feedback was attenuated, making HepG2R more sensitive to combined agents. This might be a promising instruction to overcome induced chemoresistance in cancer treatment, yet our hypothesis required further investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBEZ235 combined with HPG-PCL-PTX induced cell cycle arrest in the G2/M phase\u003c/h2\u003e \u003cp\u003eInduction of cell cycle arrest at the G2/M phase is the most prominent consequence of microtubular stabilization by PTX. To examine whether the combination treatment with HPG-PCL-PTX and BEZ235 enhanced this effect on the cell cycle of HCC, we carried out a cell cycle analysis by fow cytometry. The results showed that the combination treatment remarkably all increased the G2/M cell population in HepG2 and HepG2R after 24 hours treatment, compared to the single drug treatments and the untreated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). We also observed a increased the sub-G1 (apoptotic) cell population after 24 hours treatment, however remarkable decrease in the G1 phase. It revealed that HPG-PCL-PTX combined with BEZ235 promoted apoptosis of HCC cells. CyclinB1 and activation of the cyclin-dependent kinase 1(CDK1)/cyclinB1 complex have been shown to regulate the cell cycle transition from G2 to the M phase. Increased expression of cyclinB1 accelerates mitosis and may lead to excessive proliferation of cells\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Thus, we examined the expression of endogenous cyclin B1 and CDK1 by western blot. The results showed that the expression of cyclin B1 and CDK1 in hepG2R cells was significantly higher than in HepG2 cells\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. However, the expression of the cyclin B1 and CDK1 were reduced in HCC cell lines when treated with HPG-PCL-PTX plus BEZ235(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These data indicated that HPG-PCL-PTX and BEZ235 in combination enhanced cells arrest at G2/M phase through the down-regulation of the cyclin B1 and CDK1, inhibited cell proliferation and promoted cell apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eBEZ235 combined with HPG-PCL-PTX induced apoptosis in HCC cells\u003c/h2\u003e \u003cp\u003eJC-1 assay was used to detect the mitochondrial dysfunction of the cells\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. To investigate the effect of apoptosis induced by HPG-PCL-PTX combined with BEZ235 in HCC cells, flow cytometry analysis and JC-1 staining for mitochondrial membrane potential (ΔΨm) were performed. In flow cytometry analysis, the green/red fluorescence intensity ratio of HPG-PCL-PTX group(HepG2:27.5%, HepG2R:29.9%) was significantly higher than HPG-PCL group(HepG2:7.8%, HepG2R:4.3%), PTX group(HepG2:15.6%, HepG2R:12%), BEZ235 group(HepG2:13.4%, HepG2R:7.7%), PTX\u0026thinsp;+\u0026thinsp;BBEZ235 group(HepG2:17.2%, HepG2R:20.6%) and control group (HepG2:8.2%, HepG2R:5.2%) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). While HPG-PCL-PTX combined with BEZ235, the green/red fluorescence intensity ratio was significantly higher than HPG-PCL-PTX group (HepG2: 70.1%, HepG2R: 49.6%)(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). JC-1 staining showed the green fuorescence intensity of HCC cells treated with HPG-PCL-PTX monotherapy increased only slightly. Interestingly, when combining with BEZ235, the green fuorescence intensity was signifcantly enhanced(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). These results suggested that HPG-PCL-PTX combined with BEZ235 induces apoptosis of HCC cells by changing mitochondrial membrane potential.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePTX-mediated mitotic arrest causes apoptosis, however, resistance to apoptosis frequently arises during long-term treatment\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. So, We assayed the effect of PTX and BEZ235 combination treatment on cell death using the Annexin V FITC/PI apoptosis kit. In flow cytometry analysis, HPG-PCL-PTX (HepG2:8%, HepG2R:6.1%) caused some apoptotic cell death and slight early apoptosis after 24h treatment, was sightly higher than HPG-PCL group(HepG2:2.6%, HepG2R:2.6%), PTX\u0026thinsp;+\u0026thinsp;BEZ235 group(HepG2:5.1%, HepG2R:5.2%), PTX group(HepG2:3.0%, HepG2R:2.7%), BEZ235 group(HepG2:3.4%, HepG2R:3%) and control group(HepG2:3.3%, HepG2R:2.7%). Whereas the combinatorial application of HPG-PCL-PTX and BEZ235 produced a greater effect, inducring a higher apoptotic rate in HCC cells(HepG2:15.5%, HepG2R:15%). The apoptotic rate of HepG2 cells were slighterly higher than in HepG2R cells(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Annexin V FITC/PI staining showed that the apoptosis rate of HCC cells treated with HPG-PCL-PTX monotherapy increased only slightly. However, when combined with BEZ235, the apoptosis rate was signifcantly enhanced(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Molecular investigations indicated that the expression level of pro-apoptotic proteins (caspase-9, caspase-3, caspase-7, Bad, Bax, Bim, Cyt C and Apaf-1) was increase and anti-apoptosis(Bcl-2) was decreased after HPG-PCL-PTX plus BEZ235 treated to HCC cells(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). These results proved that HPG-PCL-PTX plus BEZ235 can promote the apoptosis of HCC cells and restore most of the sensitivity of these cells to PTX.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHPG-PCL-PTX combined with BEZ235 enhanced the anti-tumor effect\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ein vivo\u003c/span\u003e \u003cb\u003eby inhibiting PI3K/mTOR and MAPK pathway\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAnti-tumor effects of HPG-PCL-PTX combined with BEZ235 in vivo were evaluated also in a murine xenograft model using HepG2R cells. Tumor tissues of each group were isolated and collected after 30-day treatment of tumor-bearing mice. The tumor growth of animals treated HPG-PCL-PTX combined with BEZ235 group was significantly inhibited compared with that in the HPG-PCL-PTX, BEZ235 and control groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The molecular mechanism of the anti-tumor activity of HPG-PCL-PTX combined with BEZ235 was further examined by WB analysis of protein lysates from HepG2R xenografts. The results showed that HPG-PCL-PTX combined with BEZ235 group significantly reduced the phosphorylation levels of PI3K, Akt, S6K and Erk compared with levels in the control, paclitaxel and BEZ235 groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). There was no significant change in the body weight of the mice during the entire treatment period.(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Thus, we concluded that BEZ235 significantly enhanced the antitumor effect of paclitaxel in HCC \u003cem\u003ein vivo\u003c/em\u003e, at least in part by inhibiting the PI3K/Akt/mTOR pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this work, we developed the HPG-PCL deliver PTX, than combined with BEZ235 for activity against the HepG2 and HepG2R cells. At the cytological level, BEZ235 and HPG-PCL-PTX in combination had significantly more effect on proliferation and apoptosis than did either drug alone. Similarly, in vivo, HPG-PCL-PTX combined with BEZ235 treatment had a greater inhibitory effect on tumor growth than did either drug alone.\u003c/p\u003e \u003cp\u003eDespite its potent and extensive antitumor activity, the efficacy of PTX is limited by resistance\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Thus, new strategies are needed to supress the progress of HCC. The mechanism of HCC resistance to treatment is complicated: the PI3K/Akt/mTOR pathway is activated in 30\u0026ndash;50% of HCC\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, and the MAPK pathway is also activated in HCC\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Activated PI3K/Akt/mTOR and MAPK induces cell survival and proliferation and induces HCC resistance\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Our study also confirmed that PI3K/Akt/mTOR and MAPK pathways in HCC cells were significantly activated after PTX resistance, promoting cell proliferation and inhibiting apoptosis.\u003c/p\u003e \u003cp\u003ePresumably, HCC cells acquired resistance by promoting cell cycle progression and inhibiting apoptosis. CyclinB1 is a crucial regulator in G2/M transition, whose upregulation has been identified to confer resistance to PTX\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Consistently, increased cyclinB1 was observed in our established PTX-resistant cells. To further evaluate functional status of G2/M regulate factors, we also compared their expression level of parental cells and their resistant cells. The cyclinB1 accumulation and CDK1 de-phosphorylation mediating G2/M block\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. According to our results, PTX-resistant cells had reduced responses to PTX, giving a hint that the acquisition of PTX resistance attenuated PTX-mediated inability of mitotic spindle formation.\u003c/p\u003e \u003cp\u003eFurthermore, apoptosis triggered by mitotic arrest is one a major route for PTX-induced antitumor effects\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. PTX activates caspase-dependent apoptotic pathways after inducing G2/M arrest in prostate cancer cells\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Given that mitosis stalling can initiate the apoptotic pathways, we tested whether apoptotic resistance occurred in HCC cells. Our research confirms that HepG2R cells had greater anti-apoptotic capacity, as indicated by reduced caspase-9, caspase-3 ,caspase7, Bim and Bax than in HepG2 cells\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Apoptotic induction has been considered as a candidate to treat PTX-resistant cancers\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, as we found that impact of PTX on these pro-apoptotic proteins was less effective in resistant cells than in parental, targeting PI3K/Akt/mTOR might be a more efficient option against PTX-resistant cancer than apoptotic induction.\u003c/p\u003e \u003cp\u003eThe free PTX in vitro did not decrease the phosphorylation levels of p-Akt, c-Raf, MEK and ERK kinases in HepG2 or HepG2R cells, which may be due to a stress response or self-protection mechanism. In other words, PTX stimulated the activation of the PI3K/Akt/mTOR pathway in HepG2 and HepG2R cells. These results confirmed previous findings that the classical pathway in HCC cells may crosstalk to stimulate activation of other signaling pathways\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. In fact, the stimulation of Hepatocyte growth factor secretion in HCC cells by PTX promoted the phosphorylation of S6K and 4EBP1, which further stimulated the activation of the PI3K/Akt/mTOR pathway, thus supporting the hypothesis that the free PTX treatment may not effective against HCC. Therefore we prepared HPG-PCL nanoparticles loaded with PTX (HPG-PCL-PTX). When the polymer complexes passed through the circulatory system, PTX were delivered to HCC tissues through the high permeability and retention effect of the nanoparticles. Animal experiments show that treatment with HPG-PCL-PTX combined with BEZ235 significantly inhibited tumor growth; average tumor volume was significantly smaller than other treatment groups. Meanwhile, the body weight of the mice treated with HPG-PCL-PTX combination of BEZ235 group was not significantly decreased; this finding suggests that HPG-PCL-PTX combined with BEZ235 have advantages in drug delivery less delivery of the drug to non-targeted tissues, with attendant less toxicity and fewer side effects.\u003c/p\u003e \u003cp\u003eCorrespondingly, HPG-PCL-PTX combined with BEZ235 were enriched in HepG2R cells, this result would be expected to improve the anti-tumor effectiveness of the drugs. In fact, increasing research has found that Ras/Raf/MAPK and PI3K/Akt/mTOR are the main signaling pathways activated in HCC cells\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. HPG-PCL-PTX combined with BEZ235 can reduce proliferation and induce apoptosis in HCC cell lines; in xenograft mouse models enhanced tumor necrosis\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Our results corroborate these findings that during the treatment of HCC, PTX alone inhibits the Ras/Raf/MAPK signaling pathway but has limited efficacy and is likely to cause desensitization of HCC to PTX. HPG-PCL-PTX combined with BEZ235 will increase the HCC cells inhibition effect of PTX, especially for PTX-resistant HCC.\u003c/p\u003e \u003cp\u003eIn summary, our results document that HPG-PCL-PTX combined with BEZ235 have significant anti-HCC effects in vitro and in vivo, especially in PTX-resistant HCC. Thus, BEZ235 combined with PTX of transportation via nanocarriers is a promising treatment for controlling HCC, especially for PTX-resistant HCC.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eReagents and antibodies\u003c/h2\u003e \u003cp\u003ePTX, 5,5\u0026rsquo;,6,6\u0026rsquo;-tetrachloro-1,1\u0026rsquo;,3,3\u0026rsquo;-tetraethyl-benzimidazolyl carbocyanine iodide (JC-1), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), Annexin V-FITC/PI Kit and DAPI were purchased from Sigma-Aldrich (St. Louis, MO, USA). BEZ235(Dactolisib) was obtained from MedChem Express (Monmouth Junction, NJ, USA). RPMI-1640 was obtained from Hyclone (Salt Lake City, UT, USA). FBS was purchased from Hangzhou Sijiqing Company. PI3K Antibody Kit (9655#), Akt Antibody Kit (9916#), mTOR Antibody Kit (9964#), Bcl-2 Family Antibody Kit (9942#), Apoptosis Antibody Kit (9915#), secondary goat anti-rabbit and anti-mouse antibodies were purchased from Cell Signal Technology (Danvers, MA, USA). Cyclin B1 and Cyclin-dependent-kinase (CDK1) were purchased from Abcam Biological Technology (USA). β-actin was obtained from Cell Signal Technology (Danvers, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and cell culture\u003c/h2\u003e \u003cp\u003eHuman HepG2 cell line was bought from the American Type Culture Collection (Manassas, VA, USA). HepG2R, a cell line resistant to paclitaxel by HepG2 cells. The process of inducd HepG2R was as follows: When the cells were in the logarithmic growth phase, added PTX with a lower concentration for 24h, then performed cell inheritance, and repeatedly stimulated with this concentration until it was stable. Increasing the concentration of PTX continued to stimulate to a stable state. When the resistant strain treated with PTX reached 4\u0026ndash;5 times the IC50 of the sensitive strain, the resistant strain was obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of HPG-PCL\u003c/h2\u003e \u003cp\u003eThe amphiphilic HPG derivative PCL-HPG was synthesized by anionic ring-opening polymerization as follows: First 1.5g (0.10 mmol) of dry PCL was weighed into a 50mL Schlenk flask and repeatedly evacuated several times with nitrogen gas using an anhydrous oxygen-free. Then 0.8mL of 25% potassium methanol solution was added and the temperature was gradually increased to 95\u0026deg;C while stirring. 10min later, the excess methanol was removed by re-evacuation. Subsequently, 4.5g (60.7mmol) of epichlorohydrin was added dropwise to the reaction system using a micro syringe pump under the protection of nitrogen. After the dropwise addition, the reaction was continued for 5h. At the end of the reaction, the product was dissolved with an appropriate amount of methanol and the acidified cation exchange resin was added to remove the potassium ions. The crude product was removed by rotary evaporation and lyophilised using a dialysis bag with a cut-off molecular weight of 3000 to give a clear yellow viscous solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of HPG-PCL-PTX nanoparticles\u003c/h2\u003e \u003cp\u003eWeigh 500mg of HPG-PCL in 4mL of water, 10mg of PTX in 2mL of acetone. The PTX solution was added to the HPG-PCL solution drop by drop (as the PTX solution was added, the system gradually became cloudy, and the solution was clarified by adding acetone) and stirred at room temperature overnight. The drug-loaded nanoparticles were lyophilised by dialysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003echaracterization of HPG-PCL-PTX\u003c/h2\u003e \u003cp\u003eThe average particle-diameter distribution and zeta potential of the nanospheres were tested by a Malvern Zetasizer Nano ZS (Malvern, Worcester, UK). The morphology of the NPs was observed with a transmission electron microscope (TEM, Tecnai F30, FEI Company, Hillsboro, OR, USA). The structure of the synthesized PLGA-PEG in CDCl3 was confirmed by the 1H NMR spectra (Varian Unity Inova 400 Mhz, Agilent Technologies, Inc., Santa Clara, CA, USA). To analysis drug loading content (LC%) of NPs, high-performance liquid chromatography (HPLC, LC 1200, Agilent Technologies, Santa Clara, CA, USA) was applied. Calculate LC% by the following equation : LC%=(BEZ235weight inNPs)/(totalNPsweight)\u0026times;100%\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMolecular targeted\u003c/h2\u003e \u003cp\u003eThe HPG-PCL-PTX was labeled with FITC, then co-cultured with HepG2 and HepG2R cells. The nanomedicine with unmodified targeting molecules used as a control, observe the endocytosis effect of the two cell lines on nano-drugs at 0.5h, 1h, 2h and 4h, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCell viability\u003c/h2\u003e \u003cp\u003eHepG2 and HepG2R cells (5000\u0026ndash;10000 per well) were plated into 96-well plates and incubated overnight in a 5% CO2 incubator at 37\u0026deg;C. After indicated drugs exposured for 24 h, cell viability was measured using MTT assay according to the manufacturer\u0026rsquo;s instructions. Absorbance was measured at 450 nm using a spectrophotometer. IC50 was calculated by Graphpad Prism Version 5.0 software. The resistant index (RI) was calculated utilizing the following formula: RI\u0026thinsp;=\u0026thinsp;IC50 of resistant cells/IC50 of parental cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eColony-formation assay\u003c/h2\u003e \u003cp\u003eCells were cultured into 6-well plates at a density of 1000 cells per well to adhere overnight, then the cells were treated with the drugs for 24h. After about two weeks, the cell colonies were clearly visible. The medium was aspirated, colonies were fixed in 4% paraformaldehyde for 15min and stained with 0.1% crystal violet for 30min. Cell colonies were counted, and the number cultured with drugs was compared with the control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analysis\u003c/h2\u003e \u003cp\u003ePropidium iodide (PI) was used to stain the DNA content. After drug interventions for 24 h, cells were collected, washed with PBS, and fixed with 70% precooled ethanol. Before testing, cells were washed with PBS three times, added 50\u0026micro;g/ml PI and 100\u0026micro;g/ml RNase A in the dark for 30min. Dye was removed, the cells were resuspended with PBS, followed by flow cytometry analysis (BD FACSCalibur, USA). The distribution of cells at specifific cell cycle stages was assessed with ModFit Version 3.0 software (Verity Software House, Topsham, ME).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis detection\u003c/h2\u003e \u003cp\u003eCells were seeded in 24-well plates and incubated for overnight. After drug treatment for 24h, cells were collected after digestion and centrifugation, double-stained with Annexin V-FITC(10\u0026micro;L) and PI(5\u0026micro;L) at room temperature for 20min in the dark. Cell apoptosis was detected by flow cytometry within 1 h (BD Biosciences) and analyzed apoptotic rates using the Flow Jo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial membrane potential (ΔΨm) measurement\u003c/h2\u003e \u003cp\u003eJC-1 is a sensitive probe for measuring mitochondrial membrane potential. The high/low mitochondrial membrane potential of cells treated with dugs determined red/green fuorescence intensity excited by polymer/monomer of JC-1, from which the mitochondrial apoptosis level of the cells was analyzed. Cells were cultured in 24-well plates overnight, treated with drugs for 24h, counterstained with DAPI and JC-1, examined with fluorescence microscope. Besides, cells treated with drug were stained with 10\u0026micro;g/ml JC-1 for 30min at room temperature and analyzed with flow cytometry for changes in ΔΨm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eTotal protein was extracted from cells lysed with radioim munoprecipitation bufer (RIPA, Beyotime Biotechnology, Shanghai, China) containing a protease inhibitor cocktail (Beyotime Biotechnology, Shanghai, China) and centrifuged. The BCA protein assay kit (Biosharp, Hefei, China) was used for measuring protein concentration. Soluble lysates containing about 20 \u0026micro;g proteins per sample were resolved with sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis(SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (Millipore, Sigma, USA). After blocking using 5% skim milk, membranes were probed with primary antibodies (dilutions were 1:1000) at 4\u0026deg;C overnight and secondary antibodies (1:2000) at room temperature for 1 h. Protein bands were visualized with an ECL luminescent detection kit (Thermo Fisher Scientific Waltham, MA, USA) and images were captured with a gel imager Bio-Rad (Hercules, CA, USA). Quantified with Image J Version 1.48 software (NIH, Bethesda, MD). The protein content of β-actin was used as a loading control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eXenograft studies\u003c/h2\u003e \u003cp\u003e All animal experiments were approved by the Animal Experimental Ethics Committee of Anhui University of Science and Technology and were carried out in accordance with appropriate procedures. Animal experiments were carried out in the SPF-level animal room of the Central Laboratory of Medical School Anhui University of Science and Technology (NO: AUST201810088). 100\u0026micro;l of HepG2 cell suspension (2\u0026times;10\u003csup\u003e7\u003c/sup\u003e/ml) were injected subcutaneously into the dorsal right side of 5-week-old female BALB/c nude mice (Vital River Laboratories, Beijing, China). When tumor volume reached 100 mm3, mice were randomized into five groups: control group: 100\u0026micro;l saline daily, ip; HPG-PCL-PTX group: 5mg/kg weekly, ip; BEZ235 group: 45mg/kg daily, oral treatment. (The dose of BEZ235 used \u003cem\u003ein vivo\u003c/em\u003e was based on the specifications of Selleck Chemicals); HPG-PCL-PTX\u0026thinsp;+\u0026thinsp;BEZ235 group: PTX (5mg/kg weekly, ip) plus BEZ235 (45mg/kg daily, oral treatment). Each group of rats received treatment for 28 days, and tumor size and body weight were measured every 3 days. Tumor volume was calculated according to the formula: V\u0026thinsp;=\u0026thinsp;L\u0026times;W\u003csup\u003e2\u003c/sup\u003e\u0026times;1/2 (V, volume; L, length of tumor; W, width of tumor). Mice were sacrificed after the treatment; tumor tissues were isolated; protein was extracted for WB detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll analyses were performed with SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). All experiments were repeated three times independently and analyzed by Student t-test or two-way ANOVA using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was taken as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlqahtani A, Khan Z, Alloghbi A, Said Ahmed TS, Ashraf M \u0026amp; ammouda DM. Hepatocellular Carcinoma: Molecular Mechanisms and Targeted Therapies. Medicina (Kaunas) \u003cb\u003e55\u003c/b\u003e, 526(2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang JD, Hainaut P, Gores GJ, Amadou A, Plymoth A \u0026amp; Roberts LR. A global view of hepatocellular carcinoma: trends, risk, prevention and management. Nat Rev Gastroenterol Hepatol \u003cb\u003e16\u003c/b\u003e, 589\u0026ndash;604(2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou H, Li L, Garcia Carcedo I, Xu ZP, Monteiro M \u0026amp; Gu W. Synergistic inhibition of colon cancer cell growth with nanoemulsion-loaded paclitaxel and PI3K/mTOR dual inhibitor BEZ235 through apoptosis. Int J Nanomedicine \u003cb\u003e11\u003c/b\u003e, 1947\u0026ndash;58(2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChakraborty S, Dlie ZY, Chakraborty S, Roy S, Mukherjee B, Besra SE. et al. Aptamer-Functionalized Drug Nanocarrier Improves Hepatocellular Carcinoma toward Normal by Targeting Neoplastic Hepatocytes. Mol Ther Nucleic Acids \u003cb\u003e20\u003c/b\u003e, 34\u0026ndash;49(2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanchez-Carranza JN, Gonz\u0026aacute;lez-Maya L, Razo-Hern\u0026aacute;ndez RS, Salas-Vidal E, Nolasco-Quintana NY, Clemente-Soto AF. et al. Achillin Increases Chemosensitivity to Paclitaxel, Overcoming Resistance and Enhancing Apoptosis in Human Hepatocellular Carcinoma Cell Line Resistant to Paclitaxel (Hep3B/PTX). Pharmaceutics \u003cb\u003e11\u003c/b\u003e(10), 512(2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaville MW, Lietzau J, Pluda JM, Feuerstein I, Odom J, Wilson WH. et al. Treatment of HIV-associated Kaposi's sarcoma with paclitaxel. Lancet \u003cb\u003e346\u003c/b\u003e(8966), 26 \u0026ndash; 8(1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta N, Hatoum H \u0026amp; Dy GK. First line treatment of advanced non-small-cell lung cancer - specific focus on albumin bound paclitaxel. \u003cem\u003eInt J Nanomedicine\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 209 \u0026ndash; 21(2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGan H, Chen L, Sui X, Wu B, Zou S, Li A. et al. Enhanced delivery of sorafenib with anti-GPC3 antibody-conjugated TPGS-b-PCL/Pluronic P123 polymeric nanoparticles for targeted therapy of hepatocellular carcinoma. Mater Sci Eng C Mater Biol Appl \u003cb\u003e91\u003c/b\u003e, 395\u0026ndash;403(2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu B, Liang Y, Tan Y, Xie C, Shen J, Zhang M. et al. Genistein-loaded nanoparticles of star-shaped diblock copolymer mannitol-core PLGA-TPGS for the treatment of liver cancer. Mater Sci Eng C Mater Biol Appl \u003cb\u003e59\u003c/b\u003e, 792\u0026ndash;800(2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin Q, Yang Y, Hu Q, Guo Z, Liu T, Xu J. et al. Injectable supramolecular hydrogel formed from α-cyclodextrin and PEGylated arginine-functionalized poly(l-lysine) dendron for sustained MMP-9 shRNA plasmid delivery. Acta Biomater \u003cb\u003e49\u003c/b\u003e, 456\u0026ndash;471(2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMei L, Zhang Y, Zheng Y, Tian G, Song C, Yang D. et al. A Novel Docetaxel-Loaded Poly (ε-Caprolactone)/Pluronic F68 Nanoparticle Overcoming Multidrug Resistance for Breast Cancer Treatment. Nanoscale Res Lett \u003cb\u003e4\u003c/b\u003e(12), 1530\u0026ndash;9(2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma S, Verma A, Pandey G, Mittapelly N \u0026amp; Mishra PR. Investigating the role of Pluronic-g-Cationic polyelectrolyte as functional stabilizer for nanocrystals: Impact on Paclitaxel oral bioavailability and tumor growth. \u003cem\u003eActa Biomater\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, 169 \u0026ndash; 83(2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAwasthi N, Yen PL, Schwarz MA \u0026amp; Schwarz RE. The efficacy of a novel, dual PI3K/mTOR inhibitor NVP-BEZ235 to enhance chemotherapy and antiangiogenic response in pancreatic cancer. J Cell Biochem \u003cb\u003e113\u003c/b\u003e(3), 784\u0026ndash;91(2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eByun DS, Cho K, Ryu BK, Lee MG, Park JI, Chae KS. Et al. Frequent monoallelic deletion of PTEN and its reciprocal associatioin with PIK3CA amplification in gastric carcinoma. Int J Cancer \u003cb\u003e104\u003c/b\u003e(3):318\u0026ndash;27(2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi VS, Wong CW, Chan TL, Chan AS, Zhao W, Chu KM. et al. Mutations of PIK3CA in gastric adenocarcinoma. BMC Cancer \u003cb\u003e5\u003c/b\u003e, 29(2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElsadek B \u0026amp; Kratz F. Impact of albumin on drug delivery\u0026ndash;new applications on the horizon. J Control Release \u003cb\u003e157\u003c/b\u003e(1), 4\u0026ndash;28(2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGradishar WJ, Tjulandin S, Davidson N, Shaw H, Desai N, Bhar P. et al. Phase III trial of nanoparticle albumin-bound paclitaxel compared with polyethylated castor oil-based paclitaxel in women with breast cancer. J Clin Oncol \u003cb\u003e23\u003c/b\u003e(31), 7794\u0026ndash;803(2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlanco E, Sangai T, Wu S, Hsiao A, Ruiz-Esparza GU, Gonzalez-Delgado CA. et al. Colocalized delivery of rapamycin and paclitaxel to tumors enhances synergistic targeting of the PI3K/Akt/mTOR pathway. Mol Ther \u003cb\u003e22\u003c/b\u003e(7), 1310\u0026ndash;1319(2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNazio F, Strappazzon F, Antonioli M, et al. mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6. Nat Cell Biol \u003cb\u003e15\u003c/b\u003e, 406\u0026ndash;416(2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu YY, Wu HC, Wu JE, Huang KY, Yang SC, Chen SX. Et al. The dual PI3K/mTOR inhibitor BEZ235 restricts the growth of lung cancer tumors regardless of EGFR status, as a potent accompanist in combined therapeutic regimens. J Exp Clin Cancer Res \u003cb\u003e38\u003c/b\u003e(1), 282(2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng W, Wang B, Mao W, Wang J, Zhao Y, Li Q. et al. Enhanced efficacy of histone deacetylase inhibitor panobinostat combined with dual PI3K/mTOR inhibitor BEZ235 against glioblastoma. Nagoya J Med Sci \u003cb\u003e81\u003c/b\u003e(1), 93\u0026ndash;102(2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAung W, Tsuji AB, Sudo H, Sugyo A, Ukai Y, Kouda K. et al. Combined treatment of pancreatic cancer xenograft with 90Y-ITGA6B4-mediated radioimmunotherapy and PI3K/mTOR inhibitor. World J Gastroenterol \u003cb\u003e23\u003c/b\u003e(42), 7551\u0026ndash;7562(2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen L, Jin T, Zhu K, Piao Y, Quan T, Quan C. et al. PI3K/mTOR dual inhibitor BEZ235 and histone deacetylase inhibitor Trichostatin A synergistically exert anti-tumor activity in breast cancer. Oncotarget \u003cb\u003e8\u003c/b\u003e(7), 11937\u0026ndash;11949(2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Y, Tian T, Zou J, Wang Q, Li Z, Li Y. et al. Dual PI3K/mTOR inhibitor BEZ235 exerts extensive antitumor activity in HER2-positive gastric cancer. BMC Cancer \u003cb\u003e15\u003c/b\u003e, 894(2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu B, Li A, Zhang Y, Liu X, Zhou S, Gan H. et al. Resistance of hepatocellular carcinoma to sorafenib can be overcome with co-delivery of PI3K/mTOR inhibitor BEZ235 and sorafenib in nanoparticles. Expert Opin Drug Deliv \u003cb\u003e17\u003c/b\u003e(4), 573\u0026ndash;587(2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen D, Lin X, Zhang C, Liu Z, Chen Z, Li Z. et al. Dual PI3K/mTOR inhibitor BEZ235 as a promising therapeutic strategy against paclitaxel-resistant gastric cancer via targeting PI3K/Akt/mTOR pathway. Cell Death Dis \u003cb\u003e9\u003c/b\u003e(2), 123(2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Xie C, Li A, Zhang Y, Liu X, Zhou S. et al. BEZ235 enhances chemosensitivity of paclitaxel in hepatocellular carcinoma through inhibiting the PI3K/Akt/mTOR pathway. Am J Transl Res \u003cb\u003e11\u003c/b\u003e(12), 7255\u0026ndash;7271(2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang Z, Shi G, Jin J, Guo H, Guo X, Luo F. et al. Dual PI3K/mTOR inhibitor NVP-BEZ235-induced apoptosis of hepatocellular carcinoma cell lines is enhanced by inhibitors of autophagy. Int J Mol Med \u003cb\u003e31\u003c/b\u003e(6), 1449\u0026ndash;56(2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen L, Shan YS, Hu HM, Price TJ, Sirohi B, Yeh KH. et al. Management of gastric cancer in Asia: resource-stratified guidelines. Lancet Oncol \u003cb\u003e14\u003c/b\u003e(12), e535-47(2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCervello M, Emma MR, Augello G, Cusimano A, Giannitrapani L, Soresi M. et al. New landscapes and horizons in hepatocellular carcinoma therapy. Aging (Albany NY) \u003cb\u003e12\u003c/b\u003e(3), 3053\u0026ndash;3094(2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoon H \u0026amp; Ro SW. MAPK/ERK Signaling Pathway in Hepatocellular Carcinoma. Cancers (Basel) \u003cb\u003e13\u003c/b\u003e(12), 3026(2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBidkhori G, Benfeitas R, Klevstig M, Zhang C, Nielsen J, Uhlen M. et al. Metabolic network-based stratification of hepatocellular carcinoma reveals three distinct tumor subtypes. Proc Natl Acad Sci U S A \u003cb\u003e115\u003c/b\u003e(50), E11874-E11883(2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWani NA, Zhang B, Teng KY, Barajas JM, Motiwala T, Hu P. et al. Reprograming of Glucose Metabolism by Zerumbone Suppresses Hepatocarcinogenesis. Mol Cancer Res \u003cb\u003e16\u003c/b\u003e(2), 256\u0026ndash;268(2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOu Y, Ma L, Ma L, Huang Z, Zhou W, Zhao C. et al. Overexpression of cyclin B1 antagonizes chemotherapeutic-induced apoptosis through PTEN/Akt pathway in human esophageal squamous cell carcinoma cells. Cancer Biol Ther \u003cb\u003e14\u003c/b\u003e(1), 45\u0026ndash;55(2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan J, Kr\u0026auml;mer A, Matthess Y, Yan R, Sp\u0026auml;nkuch B, G\u0026auml;tje R. et al. Stable gene silencing of cyclin B1 in tumor cells increases susceptibility to taxol and leads to growth arrest in vivo. Oncogene \u003cb\u003e25\u003c/b\u003e(12), 1753\u0026ndash;62(2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang WL, Yu CC, Chen CS \u0026amp; Guh JH. Tubulin-binding agents down-regulate matrix metalloproteinase-2 and \u0026ndash; 9 in human hormone-refractory prostate cancer cells\u0026ndash;a critical role of Cdk1 in mitotic entry. Biochem Pharmacol \u003cb\u003e94\u003c/b\u003e(1), 12\u0026ndash;21(2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiovinazzi S, Bellapu D, Morozov VM \u0026amp; Ishov AM. Targeting mitotic exit with hyperthermia or APC/C inhibition to increase paclitaxel efficacy. Cell Cycle \u003cb\u003e12\u003c/b\u003e(16), 2598\u0026ndash;607(2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarley ME, Allan LA, Sanderson HS \u0026amp; Clarke PR. Phosphorylation of Mcl-1 by CDK1-cyclin B1 initiates its Cdc20-dependent destruction during mitotic arrest. EMBO J \u003cb\u003e29\u003c/b\u003e(14), 2407\u0026ndash;20(2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang C, Huang SB, Yang MC, Lin YT, Chu IH, Shen YN. et al. Combining paclitaxel with ABT-263 has a synergistic effect on paclitaxel resistant prostate cancer cells. PLoS One \u003cb\u003e10\u003c/b\u003e(3), e0120913(2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuran GE, Wang YC, Moisan F, Francisco EB \u0026amp; Sikic BI. Decreased levels of baseline and drug-induced tubulin polymerisation are hallmarks of resistance to taxanes in ovarian cancer cells and are associated with epithelial-to-mesenchymal transition. Br J Cancer \u003cb\u003e116\u003c/b\u003e(10), 1318\u0026ndash;1328(2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBae T, Weon KY, Lee JW, Eum KH, Kim S \u0026amp; Choi JW. Restoration of paclitaxel resistance by CDK1 intervention in drug-resistant ovarian cancer. Carcinogenesis \u003cb\u003e36\u003c/b\u003e(12), 1561\u0026ndash;71(2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Li H, Ren Y, Zou S, Fang W, Jiang X. et al. Targeting HDAC with a novel inhibitor effectively reverses paclitaxel resistance in non-small cell lung cancer via multiple mechanisms. Cell Death Dis \u003cb\u003e7\u003c/b\u003e(1), e2063(2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEbrahim HM, El-Rouby MN, Morsy ME, Said MM \u0026amp; Ezz MK. The Synergistic Cytotoxic Effect of Laser-Irradiated Gold Nanoparticles and Sorafenib Against the Growth of a Human Hepatocellular Carcinoma Cell Line. Asian Pac J Cancer Prev \u003cb\u003e20\u003c/b\u003e(11), 3369\u0026ndash;3376(2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToh TB, Lim JJ, Hooi L, Rashid MBMA \u0026amp; Chow EK. Targeting Jak/Stat pathway as a therapeutic strategy against SP/CD44 + tumorigenic cells in Akt/β-catenin-driven hepatocellular carcinoma. J Hepatol \u003cb\u003e72\u003c/b\u003e(1), 104\u0026ndash;118(2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGedaly R, Angulo P, Hundley J, Daily MF, Chen C, Koch A. et al. PI-103 and sorafenib inhibit hepatocellular carcinoma cell proliferation by blocking Ras/Raf/MAPK and PI3K/AKT/mTOR pathways. Anticancer Res \u003cb\u003e30\u003c/b\u003e(12), 4951\u0026ndash;8(2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRimassa L, Pressiani T \u0026amp; Merle P. Systemic Treatment Options in Hepatocellular Carcinoma. Liver Cancer \u003cb\u003e8\u003c/b\u003e(6), 427\u0026ndash;446(2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis work was supported by Wu Jieping Medical Foundation Clinical Research Special Fund(no. 320.6750.2022-02-2).\u003c/p\u003e\n\u003cp\u003eConflict of interest statement\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003eAuthor contribution statement\u003c/p\u003e\n\u003cp\u003eXiaolong Tang performed study concept and design; Xueke Liu performed development of methodology and writing, review and revision of the paper; Yingping Wu, Wei Xu, Lele Li provided acquisition, analysis and interpretation of data, and statistical analysis; Xiaoping Xia provided technical and material support. All authors read and approved the final paper.\u003c/p\u003e\n\u003cp\u003eEthics statement\u003c/p\u003e\n\u003cp\u003eThe animal experiments were approved by the Animal Experimental Ethics Committee of Anhui University of Science and Technology and were carried out in the SPF-level animal room of the Central Laboratory of Medical School Anhui University of Science and Technology (NO: AUST201810088).\u003c/p\u003e\n\u003cp\u003eData availability statement\u003c/p\u003e\n\u003cp\u003eAll data and materials are available without restriction.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"28ebb679-0feb-4669-bc30-9bf2642a03f8","identifier":"10.13039/100007452","name":"Wu Jieping Medical Foundation","awardNumber":"no. 320.6750.2022-02-2","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Fourth Affiliated Hospital, College of Medicine, Zhejiang University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HPG-PCL-PTX, BEZ235, PI3K/Akt/mTOR, HCC, resistance","lastPublishedDoi":"10.21203/rs.3.rs-1793410/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1793410/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHepatocellular carcinoma (HCC) is the sixth most common cancer in the global. It could be a potential resistant mechanism that paclitaxel activated the PI3K/mTOR and MAPK pathway in HCC cells. So inhibiting the abnormal activation of two pathway, which may a promising targeted therapy strategy. MTT and clone formation assays were used to measure cells proliferation activity; cell cycle distribution and apoptosis were measured by flow cytometry; western blot assessed phosphorylation level of proteins associated with cell cycle, apoptosis, PI3K/mTOR and MAPK pathway; Student t test or one-way ANOVA to measure significant differences between the means.BEZ235 combined with HPG-PCL-PTX blocked the activation of PI3K/mTOR and MAPK pathway in HCC cells, effectively inhibited cells proliferation and arrested the cell cycle at the G2/M phase. The most important is that the combination of two drugs caused mitochondrial membrane potential change, induced cytochrome C release and bind to APAF-1 to initiate the caspase-9 apoptosis program, promoted HCC cell apoptosis. HPG-PCL-PTX/BEZ235 enhanced antitumor effectiveness in HepG2R cell xenograft mouse models. In vivo study suggested that BEZ235 combined with HPG-PCL-PTX enhanced effects of HPG-PCL-PTX through inhibited the PI3K/Akt/mTOR and MAPK pathway in HCC cells, supressed cells proliferation, arrested the cell cycle in a G2/M phase and promoted apoptosis. Antitumor effects of BEZ235 and HPG-PCL-PTX were also confirmed in mice bearing HepG2R tumors. Taken together, the results of this study describe a promising strategy using PTX and BEZ235 in a nanoparticle formulation for treatment of PTX-resistant HCC.\u003c/p\u003e","manuscriptTitle":"BEZ235 enhances antitumor efficacy of HPG-PCL-PTX in HCC by suppressed the PI3K/Akt/mTOR and MAPK pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-06-29 14:39:10","doi":"10.21203/rs.3.rs-1793410/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8ae0470d-80ae-4cf5-8a68-d3deeb725a8a","owner":[],"postedDate":"June 29th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":13536486,"name":"Cell Cycle \u0026 Proliferation"},{"id":13536487,"name":"Cell Communication and Signaling"},{"id":13536488,"name":"Cell Migration and Cell Adhesion"},{"id":13536489,"name":"Cell Survival and Cell Death"},{"id":13536490,"name":"Drug Delivery"}],"tags":[],"updatedAt":"2022-06-29T14:39:10+00:00","versionOfRecord":[],"versionCreatedAt":"2022-06-29 14:39:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1793410","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1793410","identity":"rs-1793410","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}