{"paper_id":"004c50bf-de23-45c8-8cc7-bd9734920f38","body_text":"Purified Cucurbitacin D leads to alterations of apoptotic and autophagic genes expression in MDA-MB-468 and MCF-7 human breast cancer cells | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Purified Cucurbitacin D leads to alterations of apoptotic and autophagic genes expression in MDA-MB-468 and MCF-7 human breast cancer cells Elham Zeinali, Seyed Jalal Zargar, Najmeh Mozdoori This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2920606/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 Breast cancer is the second most frequent malignancy worldwide. The use of plant-derived drugs in cancer therapy are widely considered in the treatment of different malignancies including breast cancer. Cucurbitacin D (Cu D) is able to induce apoptosis in cancerous cells through different signaling pathways. The aim of this study was to examine the effect of different concentrations of Cucurbitacin D on viability, death pattern as well as the expression alterations of Bcl-2 , Bax , caspase-3 , p53, Atg5 , Beclin-1 , PTEN and Akt genes in the MCF-7 (ER positive) and MDA-MB-468 (triple negative) breast cancer cell lines. Two breast cancer cell lines (MCF-7 and MDA-MB-468) were cultured and treated with different concentrations of the purified Cucurbitacin D. Anti-proliferative effects of the Cucurbitacin D on both breast cancer cell lines viability was investigated using the MTT assay. Real-time PCR was applied to evaluate the expression levels of the genes upon Cucurbitacin D therapy. Significant dose-dependent and anti-proliferative effects of the Cucurbitacin D were observed on MCF-7 and MDA-MB-468 cells after 24 h with IC50 value about 30 and 25 µM, respectively (P < 0.01). Significant changes in expression of the genes in the breast cancer lines (MCF-7 and MDA-MB-468), were observed under different concentrations of Cucurbitacin D. Our results confirmed that the Cucurbitacin D may influence breast cancer cell lines viability at specific doses and by altering the expression of these genes. The differences between the gene’s aberrations in our breast cancer cell lines propose that these genes can have a distinct role in the pathophysiology and therapy responsiveness of various subtypes of breast cancer. Breast cancer Cucurbitacin D Cell viability Apoptosis Autophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Breast cancer is the second most frequent malignancy worldwide after lung cancer and the fifth most common cause of cancer death among women (Aggarwal et al., 2020; Rafatian et al., 2021). Several factors including hormone or estrogen-responsiveness and expression of estrogen or progesterone receptors can determine prognosis of human breast cancer (Shokrollahi Barough et al., 2015). Breast cancer cell lines are extensively used for studying various breast cancers pathogenesis and developing new treatment strategies (Subik et al., 2010; Zareian et al., 2022). Breast cancer cell lines are classified according to their differences in terms of estrogen receptor (ER), progesterone receptor (PR) ERBB2, and HER2/neu expression panel. Estrogen independent or ER negative cell lines such as MDA-MB-468 isolated from high metastatic ER/PR and HER2-negative adenocarcinoma are more aggressive and higher metastatic. However, ER positive cell lines such as MCF-7 isolated from ER/PR positive and HER2-negative adenocarcinoma are less aggressive (Shokrollahi Barough et al., 2015). Compared to chemically synthesized cancer medications, plant-derived drugs used in cancer therapy are natural products with less side effects and toxicity (Lee et al., 2010). Cucurbitacins refer to a cluster of tetracyclic triterpenoids isolated from different plant families, including Cucurbitaceae (Ecballium elaterium) and Cruciferae . Cucurbitacins are known to have multiple therapeutic effects such as anti-proliferation and anti-cancer (Lee et al., 2010). Cucurbitacins consist of 17 main molecules from cucurbitacin A to cucurbitacin T, among which cucurbitacin B, D, E, I, have been known mainly due to their significant anticancer and antitumor activities (Sikander et al., 2016). Different investigations on Cucurbitacin D (Cu D) revealed its remarkable anticancer activity in many human cancer cell lines (Lee et al., 2010). It was recently reported that Cucurbitacin D is able to induce apoptosis in cancerous cells through different signaling pathways (Kim et al., 2020) . Apoptosis or programmed cell death is a fundamental event that occurs in a tightly regulated and precise manner and is particularly significant during tissue development and maturation and numerous pathological conditions (Ferhi et al., 2019; Singh et al., 2019). The Bcl-2 family, a group of about 25 genes governing mitochondrial membrane permeability with a pivotal role in the regulation of apoptosis, can be either pro-apoptotic or anti-apoptotic. These proteins have special significance since they can determine if the cell commits to apoptosis or not (Elmore, 2007). The regulation of Bcl-2 family at the transcriptional level seems to be a critical factor in the regulation of cell death (Burlacu, 2003). Bcl-2 is one of the most prominent members of anti-apoptotic proteins which are able to promote cell survival. Bax , a member of pro-apoptotic proteins, and caspase-3 , a key component of a family of proteases with prominent role in apoptotic response, are also among main regulators of apoptosis (Peña-Blanco and García-Sáez, 2018). DNA damage caused by cancer chemotherapy drugs in some cells, can lead to apoptotic death through a p53 -dependent pathway. In fact, the tumor suppressor protein p53 has a critical role in p53 -mediated apoptosis through regulation of the Bcl-2 family members such as Bcl-2 and Bax (Elmore, 2007) . Autophagy is one of the key evolutionary mechanisms mediating stress-induced metabolic adaptation and damage control and is implicated in various diseases such as cancer (Codogno and Meijer, 2005; Kroemer et al., 2010). The genes that are responsible for the regulation of autophagy are known as autophagy genes or Atgs. Atg5 is an autophagic gene which promotes autophagy and may also be involved in pro-apoptotic pathway (Yousefi and Simon, 2007). Beclin1 is the mammalian ortholog of yeast Atg6 which has a critical role in autophagosome formation. Loss of Beclin1 is tightly associated with defects in autophagy (Menon and Dhamija, 2018) . Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a dual lipid and protein phosphatase whose major substrate is phosphatidylinositol-3,4,5-triphosphate (PIP3), and downstream of which lies the PTEN/PI3K/Akt pathway (Kurose et al., 2001). Therefore, AKT is a serine–threonine kinase downstream of PTEN/PI3K, whose activity is regulated by PI3K and its activation results in the suppression of apoptosis. In addition, hyper-activated AKT can promote cell proliferation and cell growth. PTEN/PI3K/Akt pathway members aberrations are common in breast cancer and have been shown to play a distinct role in the pathogenesis of different types of breast tumors (Carnero et al., 2008). Notwithstanding, targeting this pathway will provide an effective therapeutic approach in breast cancer (Li et al., 2017). The anti-cancer effects of Cucurbitacin D on some tumors and tumor-derived cell lines have been proved. However, the anticancer effects of Cucurbitacin D on breast cancer awaits further delineation. The aim of the present study was to examine the effects of Cucurbitacin D on apoptosis as well as the expression levels of Bc-l2 , Bax , caspase-3 , p53, Atg5 , Beclin-1 , PTEN and Akt genes in the MCF-7 and MDA-MB-468 cell lines. 2. Material and Methods 2. 1. Cell Lines, Cell Culture and Treatments Two human breast cancer cell lines, MCF-7 and MDA-MB-468, were purchased from Iranian Biological Resource center (Tehran, Iran). Cell lines were cultured in DMEM/F12 medium. 2. 2. Cell Viability Assay (Cytotoxicity) Anti-proliferative effects of the Cucurbitacin D against MCF-7 and MDA-MB-468 human breast cancer cell lines and cell viability were investigated using the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), assay. Various concentrations of Cucurbitacin D in DMEM/F12 were provided (0.1–100 µM for MCF-7 and 1–70 µM for MDA-MB-468) and cells were treated for an additional overnight incubation. At the end, the absorbance of it was measured at 570 nm using an ELISA plate reader. 2. 3. RNA Extraction Total cell RNA from breast cancer cell lines was extracted using Qiagen RNeasy Mini Kit (QIAGEN, Germany), according to the manufacturer’s protocol. 2. 4. Reverse Transcription and Quantitative Real-Time PCR (RT-qPCR) For cDNA synthesis, 2 µg of isolated RNA samples were reverse-transcribed with random primer according to the manufacturer’s instructions by Easy cDNA Synthesis Kit (Cat. No. A101161, Pars Tous Biotechnology, Iran). The expression levels of the genes were analyzed based on the cycle threshold (Ct) and relative expression levels were determined as 2 −ΔΔC(t) . 2. 5. Statistical analysis Statistical analyses were carried out using GraphPad Prism 8. Comparison between groups was analyzed by Student's independent-samples t -test. Data are presented as the mean ± SD of three independent experiments and p values < 0.05 were considered statistically significant. (*: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001; ****: P ≤ 0.0001). 3. Results 3. 1. Antiproliferative activities of Cucurbitacin D Based on the articles which we took into consideration in the first place, we decided to create a serial dilution of the drug which was as low as 0.1 µM to as high as 100 µM, in MCF-7 and from 1 µM to 70 µM, in MDA-MB-468. According to the graph which was accomplished by MTT results inserted in the excel program, IC50 was calculated based on the equations of each line. In the absence of cucurbitacin D, MCF-7 cells show more than 98% viability. Upon addition of cucurbitacin D, the MCF-7 cell viability was reduced to 88%, at 0.1 µM of cucurbitacin D until it reaches 83% at 0.5 µM. Then the cell viability experiences a slight increase followed by a gradually drop till reaches 68% at 20 µM. There was a significant decrease in the cell viability after cucurbitacin D exposure, especially at high concentrations (≥ 50 µg/ml). (mean ± SD, n = 3). The same behavior was seen in MDA-MB-468 cell lines. The cell viability of MDA-MB-468 cells stands in about 99% in absence of Cucurbitacin D and subsequently decreases from 80% at 1 µM to 70% at 20 µM. A sudden drop is shown in cell viability at concentrations higher than 20 µM. The half-maximal concentrations of proliferation inhibition or IC50 which reduced cell viability by 50% after 24 hours of incubation were 30 and 25 µM for MCF-7 and MDA-MB-468 cells, respectively. These IC50s as well as the concentrations in which the percentages of viability were 30% and 80% were chosen for subsequent experiments (Fig. 1 ). These concentrations are 50 and 0.5 µM of Cu D for MCF-7 and 50 and 1 µM for MDA-MB 468 cell line. 3. 2. Effect of Cucurbitacin D on the mRNA expression profile of apoptosis related genes The expression levels of four key apoptotic genes were evaluated in MCF-7 and MDA-MB-468 cells cultured in the presence of Cucurbitacin D for 24 hours. The expression levels of Bax , Bcl-2 , caspase3 , and p53 in MCF-7 and MDA-MB-468 cell lines, treated with various concentrations of Cucurbitacin D can be seen in Figure 2. Treated MCF-7 cells exhibited a significant increased expression levels of Bcl-2 at all three Cucurbitacin D concentrations. However, no significant difference was shown in treated MCF-7 cells regarding expression profiles of two apoptotic genes, Bax and p53 , at 30 μM Cucurbitacin D ( P = 0.343 and 0.174, respectively). Interestingly, these genes, Bax and p53 , showed significant decreased expression levels at 0.5 and 50μM Cucurbitacin D (Figure 2A). MDA-MB-468 exhibited similar trend to MCF-7 cells regarding the expression levels of all four apoptotic genes so that in these cells’ expression levels of Bcl-2 had a significant increase at all three concentrations of Cucurbitacin D compared with untreated control cells. Bax and p53 genes had significant decreased expression levels at 1 and 50 μM Cucurbitacin and they failed to show any significant difference between treated and control cells at 25 μM (Figure 2B). No significant changes were observed in the expression levels of caspase-3 at all tested Cucurbitacin D concentrations in both cell lines. 3. 3. Effects of Cucurbitacin D on the mRNA expression profile of autophagic genes In the next step, the expression profiles of Atg5 and Beclin1 were measured as two important autophagic genes. In MCF-7 cell line, an increase in the concentration of Cucurbitacin D results in the down-regulation of both Atg5 and Beclin1 genes so that their expression was enhanced at low Cucurbitacin D concentrations ( P = 0.01 and >0.0001, respectively) while increased concentrations (50μM) led to a reduction in their expressions ( P = 0.0004 and 0.001, respectively). Atg5 and Beclin1 genes levels did not change remarkably at IC50 ( P = 0.16 and 0.76, respectively). Nevertheless, in MDA-MB-468 cell line autophagic genes indicated a different expression pattern and Beclin1 expression showed similar pattern to Atg5 . In this cell line, the expression levels of the autophagic genes showed significant upregulation only at 25 μM and their levels did not change at concentrations other than IC50 (Figure 3). 3. 4. Effect of Cucurbitacin D on the mRNA expression profile of cell survival genes A preliminary comparison between the relative mRNA expressions of Akt and PTEN in treated MCF-7 cells and control cells revealed that enhanced Cucurbitacin D concentration (50μM) led to a significant reduction in expression levels of Akt and increase in PTEN levels. Both MCF-7 and MDA-MB-468 cell lines demonstrated significant increased expression of PTEN at different concentrations of Cucurbitacin D (Figure 4). MCF-7 and MDA-MB-468 breast cancer cells showed significant lower expression levels of Akt only at enhanced Cucurbitacin D concentrations (50μM). 4. Discussion Unfortunately, despite improved cure rate, there is still limited success in breast cancer treatment so that more than 400,000 patients are estimated to die from this malignancy annually (Li et al., 2016). Natural therapies based on medicinal plants or traditional herbal remedies have gained increasing attention for the cancer prevention (Jafargholizadeh et al., 2018). Cucurbitacins belonging to a group of tetracyclic triterpenoids with a wide range of biological effects, are able to prevent cell growth and induce apoptosis in a wide variety of cancer cell lines (Kim et al., 2020). Since cucurbitacins were identified with emerging anti-cancer activities, such as pro-apoptosis and autophagy induction, they have been reported to be a potential candidate for various cancer therapies, including gastric, breast, ovarian, and lung (Luo et al., 2019). The underlying mechanisms of Cucurbitacin D anticancer effects in human breast cancer are still elusive. In this study, the effect of different concentrations of Cucurbitacin D on cell viability and expression levels of some genes related to the apoptosis, autophagy and cell survival was investigated in MCF-7 and MDA-MB-468 human breast cancer cell lines. MDA-MB-468 was used as an estrogen independent or ER-negative cell line which is triple negative and more aggressive and metastatic than MCF-7 utilized as an estrogen dependent or ER-positive cell line. Results of cell survival assay after 24 h of MCF-7 and MDA-MB-468 cells exposure to the Cucurbitacin D revealed that it can significantly reduce cell survival in a dose-dependent manner. The results confirmed the cytotoxic effect of Cucurbitacin D on the two breast cancer cell lines with IC50 values of 30 µM in MCF-7 and 25 µM in MDA-MB-468 cells. This is in conjunction with the results of a study by Kim et al. that showed Cucurbitacin D inhibits cell proliferation and induces G2/M phase cell cycle arrest in MDA-MB-231 cells (Kim et al., 2013). Further studies with cucurbitacin D showed that it is able to inhibit proliferation and induce apoptosis in doxorubicin-resistant breast cancer MCF-7/ADR cells (Luo et al., 2019). Besides, Kim et al. reported that Cucurbitacin D, isolated from Trichosanthes kirilowii (TKE), can induce apoptosis through activation of caspases and JNK in hepatocellular carcinoma cells (Kim et al., 2013). In fact, cucurbitacins can exert their anticancer effects through various targets (Luo et al., 2019). JAK-STAT, AKT-PKB, and MAPK pathways are some of the most common pathways involved in cancer cells, all of which are targets of the cucurbitacin family (Lee et al., 2010). Cucurbitacins can act as STAT3 inhibitors in cancer cells. JAK-STAT pathway inhibition would affect different downstream targets involved in cell growth and apoptosis. The anticancer mechanism by which cucurbitacins act in breast cancer cells is still not clear. An investigation by Tannin-Spitz et al. who exposed two breast cancer cell lines, MCF-7 and MDA-MB-231, to cucurbitacin B/E showed that these cucurbitacins can increase the STAT3 phosphorylation (Lee et al., 2010). In addition, cucurbitacin mediates apoptosis through mitochondrial-related pathway, which can be characterized by the Bcl-2 down-regulation and Bax up-regulation, that eventually leads to the caspase activation (Luo et al., 2019).The next step in this investigation was to examine whether Cucurbitacin D exert its cytotoxic effects on breast cancer cell lines through apoptotic genes or not. The Bcl-2 family proteins have special significance since they can determine if the cell commits to apoptosis or aborts the process. The main mechanism through which the Bcl-2 family of proteins act is the regulation of cytochrome c release via alteration of mitochondrial membrane permeability. Bcl-2 is one of the most prominent members of anti-apoptotic proteins (Elmore, 2007). Strikingly, both breast cancer cell lines used in the current study exhibited a significant increased expression levels of Bcl2 at all Cucurbitacin D concentrations. Bax is a well-known pro-apoptotic molecule with about 21% amino acid identity with Bcl-2 (Burlacu, 2003). Interestingly, in this investigation Bax showed significant decreased expression levels at both Cucurbitacin D concentrations higher or lower than the IC50 in MCF-7 and MDA-MB-468 breast cancer cells. Previous studies revealed that cells with high Bax/Bcl-2 ratio are more susceptible to the apoptosis due to the activation of caspases 3, 8 and 9 (Lee et al., 2012). Fascinatingly, this ratio was less than one in all the Cucurbitacin D treatments in our study. It can be concluded that none of the breast cancer cell lines treated with Cucurbitacin D are prone to apoptosis and apoptosis is not stimulated by Cucurbitacin D in these cancerous cells at least through changes in the expression levels of Bcl-2 and Bax . The tumor suppressor protein p53 has a critical role in the regulation of the Bcl-2 family. Additionally, irradiation or drugs used for cancer chemotherapy results in DNA damage in some cells, which can lead to apoptotic death through a p53 -dependent pathway. Importantly, Bcl-2 and Bax expression is shown to be regulated by the p53 tumor suppressor gene (Elmore, 2007). Notably, our data indicate that the expression level of p53 did not change at IC50 concentrations, while its expression decreased significantly at Cucurbitacin D concentrations other than IC50 in the two tested cancerous cell lines. This observation can confirm the results of Bcl2 and Bax expression showing that apoptosis is not triggered in the breast cancer cells treated with Cucurbitacin D through these two genes. Moreover, Bcl-2 and Bax expression dysregulation can be due to the deregulated expression levels of p53 . A study by Yuan et al (Lee et al., 2012). using GBM cells treated with cucurbitacin I for 48 h confirmed significant increased Bax and cleaved caspase-3 but decreased antiapoptotic proteins such as Bcl-2 and Bcl-xL in a dose-dependent manner (Jafargholizadeh et al., 2018). Further, Kim et al. reported that Cucurbitacin D induces apoptosis via activation of caspases in hepatocellular carcinoma cells (Kim et al., 2013). Caspase-3 is considered to be the most important executioner caspase whose activity lead to the apoptosis (Elmore, 2007). Noticeably, MCF-7 and MDA-MB-468 cells showed no significant changes in the expression levels of caspase-3 at all tested Cucurbitacin D concentrations. Analysis of the expression levels of four important apoptotic genes, including BCl-2 , Bax , p53 and caspase-3 and considering the role of Cucurbitacin D in apoptosis indicate that Cucurbitacin D exerts its cytotoxic effect on breast cancer cells through pathways other than the caspase-dependent cell death. So, the most important cell death mechanism in these treatments is not canonical apoptosis. One possibility could be that Cucurbitacin D may affect breast cancer cells viability through triggering non-canonical apoptosis which is independent of caspases. In other words, Cucurbitacin D could limit caspase-dependent apoptosis while it induces non-canonical apoptosis (Jafargholizadeh et al., 2018). Another possibility is that Cucurbitacin D might induce apoptosis mainly through the dysregulation of other members of the Bcl-2 family. It goes without saying that further investigations are required to throw further light on the effects of Cucurbitacin D on breast cancer cells apoptosis and its related genes. Despite of the fact that autophagy is a survival mechanism, some changes in autophagic activity can lead to the cell death. Autophagy has been reported to have a key role in tumor suppression so that reduced autophagic activity can be found in some cancers and sufficient autophagy is vital for tumor suppression (Yousefi and Simon, 2007). It has been documented that Cu E, Cucurbitacin D, and Cu I are capable of inducing autophagy in different cancer cell lines (Niu et al., 2016). It was previously reported that Cu E and Cu I-induced autophagy is dependent on ATG5 and Beclin 1 expression (Zha et al., 2015). Beclin1 is necessary for autophagic pathway. The functional and structural interaction between Beclin1 and Bcl-2 is a key event in the considerable crosstalk between autophagy and apoptosis and the regulation of autophagy (Codogno and Meijer, 2005; Nikoletopoulou et al., 2013). The p53 deacetylation can induce autophagy by reducing the interaction of Beclin-1 and Bcl-2. Atg5 is another well-known autophagic gene which is critical for the formation of autophagosomes and regulation of autophagic cell death. Noteworthy, caspase-mediated cleavage of Beclin1 and ATG5 can switch autophagy to apoptosis (Nikoletopoulou et al., 2013). Our results show that in MCF-7 cell line, an increase in the concentration of Cucurbitacin D results in the down-regulation of both Beclin1 and Atg5 autophagic genes expression levels. So, it can be suggested that at Cucurbitacin D concentrations lower than IC50 (30 µM), MCF-7 cells are more prone to autophagy, while Cucurbitacin D concentration of 50 µM does not exert cytotoxic effects on MCF-7 cells through autophagy. Remarkably, these two autophagic genes expression analysis indicated that the mechanism through which Cucurbitacin D acts in MDA-MB-468 breast cancer cells is completely different from that in the MCF-7 cells. MDA-MB-468 cells showed significant increased expression levels of both Beclin1 and Atg5 only at concentration of 25 µM (IC50). The expression levels of these genes did not change significantly at Cucurbitacin D concentrations less or higher than IC50 in these cells. It can be concluded that autophagy is triggered in MDA-MB-468 at IC50 of Cucurbitacin D. To further investigate the mechanism by which Cucurbitacin D affects breast cancer cells viability, in the next step we evaluated the expression levels of two prominent cell survival related genes. PTEN/PI3K/Akt pathway plays a vital role in breast carcinogenesis so that its aberrations are common in this cancer. The PTEN/PI3K/Akt pathway mutational aberrations are markedly different among the different breast cancer subtypes and are most common in hormone receptor–positive breast tumors. PTEN loss was reported to be associated with adverse outcomes in breast cancer (Stemke-Hale et al., 2008). PTEN has also been reported to be directly associated with p53 through increasing protein levels, stability and transcriptional activity of p53 (Carnero et al., 2008). In addition, previous investigations showed that PTEN activation might act as an upstream molecule of autophagy so that its loss of function can cause a strong inhibition of the formation and maturation of autophagosomes (Aquila et al., 2020). Ectopic expression of PTEN results in cell death in the glioma and breast cancer cell lines (Kurose et al., 2001). Noticeably, both MCF-7 and MDA-MB-468 breast cancer cell lines exhibited a significant increased expression levels of PTEN after 24h treatment with different Cucurbitacin D concentrations. A recent study by Niu. et al revealed that Cu B inhibited SH-SY5Y cells proliferation through upregulation of PTEN (Niu et al., 2016). PTEN indirectly inhibits Akt phosphorylation which results in decreased Akt activity, the kinase downstream of PTEN/PI3K/Akt (Carnero et al., 2008). This kinase plays an essential role in the PTEN/PI3K/Akt pathway. Loss of PTEN function can lead to the constitutive activation of Akt (Sos et al., 2009). The activation of serine–threonine kinase Akt stimulates cell cycle progression and cell survival in a wide variety of cells, including cancer cells (Carnero et al., 2008). AKT-targeted therapies are being introduced into trials (Stemke-Hale et al., 2008). Noteworthy, our observations showed that Akt expression level decreased in both breast cancer cell lines after treatment with all concentrations of Cucurbitacin D. However, this decrease was only significant at the concentrations higher than IC50. PTEN suppress breast cancer growth and can cause cell cycle arrest at G1 (Paramio et al., 1999; Yu et al., 2021). Cytotoxic effects of Cucurbitacin D on breast cancer cells may involve some mechanisms including the PARP cleavage, MAPK pathway, decreasing pSTAT3 and JAK3 levels, expression of active caspase-3, and decreasing STAT3 downstream targets, even up-regulation of PTEN (Aquila et al., 2020) which needs to be further investigated. According to the results of the current investigation, it seems that Cucurbitacin D affects different breast cancer cells through different mechanisms at different concentrations so that it may induce autophagy at a particular concentration in a particular cell line while it induces apoptosis at other concentrations in another one (Jafargholizadeh et al., 2018). Besides, differences in the gene’s expression levels under the Cucurbitacin D treatment, observed in MCF-7 and MDA-MB-468 cell lines can be mainly due to the origin, genetic and macromolecular structure differences between these cell lines. Therefore, as it was previously pointed out by Raikhlin-Eisenkraft et al., the toxicity of cucurbitacins can be affected by many factors. The discrepancy between our results and previous studies can be partly explained by the extent of purity as well as the type of solvents used for elution and dilution of Cucurbitacin D (Lee et al., 2010). According to the studies performed by Naoki Wakimoto et al there was no significant connection between the sensitivity to growth inhibition by Cucurbitacin B and molecular characteristics such as ER, Her-2/neo or p53 status. This might be an effective treatment against triple negative breast cancer (Wakimoto et al., 2008). Furthermore, Tehila Tannin-Spitz and et al reported that cucurbitacin glucosides isolated from Citrullus colocynthis can have anti-proliferative activity on both estrogen-dependent and estrogen-independent human breast cancer cells by causing G2/M phase arrest. It is reasonable to hypothesize that cucurbitacin glucosides can be useful for chemotherapy of both estrogen-dependent and independent breast cancers (Tannin-Spitz et al., 2007). However, the differences between the gene’s aberrations in our breast cancer cell lines propose that these genes may have a distinct role in the pathophysiology and therapy responsiveness of various subtypes of breast cancer. 5. Conclusion This study provides molecular insight into the effects of Cucurbitacin D on breast cancer. We examined the effects of different concentrations of Cucurbitacin D on apoptosis as well as the expression levels of autophagic and cell survival genes in the MCF-7 and MDA-MB-468 breast cancer cell lines. The results of the cell viability assay confirmed the cytotoxic effect of Cucurbitacin D on the two breast cancer cell lines. In both cell lines apoptosis is not induced by Cucurbitacin D through canonical pathway. Non-canonical apoptosis pathways or dysregulation of other the Bcl-2 family members should be evaluated for further studies. Cucurbitacin D also triggers autophagy in MDA-MB-468 and MCF-7 in various concentrations (in IC50 and concentration lower than IC50; respectively). To get a more accurate conclusion, protein assays should be performed for key genes in the autophagy and apoptosis pathways. Declarations Ethical Approval This declaration is “not applicable”, because it is necessary for human and/ or animal studies. Competing interests The authors declare no conflict of interest. Authors' contributions Elham Zeinali has worked on the experimental part. Seyed Jalal Zargar has supervised this research and he has written it. Najmeh Mozdoori has contributed in writing this paper. Funding This research was funded by Iran National Science Foundation (INSF), Grant No. 90004329. We thank the Research Council of the University of Tehran for their financial support of this research. Availability of data and materials The data has been defined in this article and it doesn’t have any supplementary data. References Aggarwal, V., Priyanka, K., Tuli, H.S., 2020. Emergence of Circulating MicroRNAs in Breast Cancer as Diagnostic and Therapeutic Efficacy Biomarkers. Mol. 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Oncotarget 8, 20252–20265. https://doi.org/10.18632/ONCOTARGET.15690 Luo, H., Vong, C.T., Chen, H., Gao, Y., Lyu, P., Qiu, L., Zhao, M., Liu, Q., Cheng, Z., Zou, J., Yao, P., Gao, C., Wei, J., Ung, C.O.L., Wang, S., Zhong, Z., Wang, Y., 2019. Naturally occurring anti-cancer compounds: shining from Chinese herbal medicine. Chin. Med. 14. https://doi.org/10.1186/S13020-019-0270-9 Menon, M.B., Dhamija, S., 2018. Beclin 1 Phosphorylation - at the Center of Autophagy Regulation. Front. cell Dev. Biol. 6. https://doi.org/10.3389/FCELL.2018.00137 Nikoletopoulou, V., Markaki, M., Palikaras, K., Tavernarakis, N., 2013. Crosstalk between apoptosis, necrosis and autophagy. Biochim. Biophys. Acta 1833, 3448–3459. https://doi.org/10.1016/J.BBAMCR.2013.06.001 Niu, Y., Sun, W., Lu, J.J., Ma, D.L., Leung, C.H., Pei, L., Chen, X., 2016. PTEN activation by DNA damage induces protective autophagy in response to Cucurbitacin B in hepatocellular carcinoma cells. Oxid. Med. Cell. Longev. 2016. https://doi.org/10.1155/2016/4313204 Paramio, J.M., Navarro, M., Segrelles, C., Gómez-Casero, E., Jorcano, J.L., 1999. PTEN tumour suppressor is linked to the cell cycle control through the retinoblastoma protein. Oncogene 18, 7462–7468. https://doi.org/10.1038/sj.onc.1203151 Peña-Blanco, A., García-Sáez, A.J., 2018. Bax, Bak and beyond - mitochondrial performance in apoptosis. FEBS J. 285, 416–431. https://doi.org/10.1111/FEBS.14186 Rafatian, G., Zargar, S.J., Safarian, S., Sadjadpour, S., Mozdoori, N., 2021. LaF3: Tb3+ nanoparticles show adaptability to targeted therapy for a safer cancer cell treatment. Chem. Pap. 2021 7511 75, 5793–5801. https://doi.org/10.1007/S11696-021-01750-4 Shokrollahi Barough, M., Hasanzadeh, H., Barati, M., Pak, F., Kokhaei, P., Rezaei-Tavirani, M., 2015. Apoptosis/Necrosis Induction by Ultraviolet, in ER Positive and ER Negative Breast Cancer Cell Lines. Iran. J. Cancer Prev. 8. https://doi.org/10.17795/IJCP-4193 Sikander, M., Hafeez, B. Bin, Malik, S., Alsayari, A., Halaweish, F.T., Yallapu, M.M., Chauhan, S.C., Jaggi, M., 2016. Cucurbitacin D exhibits potent anti-cancer activity in cervical cancer. Sci. Reports 2016 61 6, 1–13. https://doi.org/10.1038/srep36594 Singh, R., Letai, A., Sarosiek, K., 2019. Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nat. Rev. Mol. Cell Biol. 20, 175–193. https://doi.org/10.1038/S41580-018-0089-8 Sos, M.L., Koker, M., Weir, B.A., Heynck, S., Rabinovsky, R., Zander, T., Seeger, J.M., Weiss, J., Fischer, F., Frommolt, P., Michel, K., Peifer, M., Mermel, C., Girard, L., Peyton, M., Gazdar, A.F., Minna, J.D., Garraway, L.A., Kashkar, H., Pao, W., Meyerson, M., Thomas, R.K., 2009. PTEN loss contributes to erlotinib resistance in EGFR-mutant lung cancer by activation of Akt and EGFR. Cancer Res. 69, 3256–3261. https://doi.org/10.1158/0008-5472.CAN-08-4055 Stemke-Hale, K., Gonzalez-Angulo, A.M., Lluch, A., Neve, R.M., Kuo, W.L., Davies, M., Carey, M., Hu, Z., Guan, Y., Sahin, A., Symmans, W.F., Pusztai, L., Nolden, L.K., Horlings, H., Berns, K., Hung, M.C., Van De Vijver, M.J., Valero, V., Gray, J.W., Bernards, R., Mills, G.B., Hennessy, B.T., 2008. An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer. Cancer Res. 68, 6084–6091. https://doi.org/10.1158/0008-5472.CAN-07-6854 Subik, K., Lee, J.F., Baxter, L., Strzepek, T., Costello, D., Crowley, P., Xing, L., Hung, M.C., Bonfiglio, T., Hicks, D.G., Tang, P., 2010. The expression patterns of ER, PR, HER2, CK5/6, EGFR, KI-67 and AR by immunohistochemical analysis in breast cancer cell lines. Breast Cancer Basic Clin. Res. 4, 35–41. https://doi.org/10.1177/117822341000400004 Tannin-Spitz, T., Grossman, S., Dovrat, S., Gottlieb, H.E., Bergman, M., 2007. Growth inhibitory activity of cucurbitacin glucosides isolated from Citrullus colocynthis on human breast cancer cells. Biochem. Pharmacol. 73, 56–67. https://doi.org/10.1016/J.BCP.2006.09.012 Wakimoto, N., Yin, D., O’Kelly, J., Haritunians, T., Karlan, B., Said, J., Xing, H., Koeffler, H.P., 2008. Cucurbitacin B has a potent antiproliferative effect on breast cancer cells in vitro and in vivo. Cancer Sci. 99, 1793–1797. https://doi.org/10.1111/J.1349-7006.2008.00899.X Yousefi, S., Simon, H.U., 2007. Apoptosis regulation by autophagy gene 5. Crit. Rev. Oncol. Hematol. 63, 241–244. https://doi.org/10.1016/J.CRITREVONC.2007.06.005 Yu, C., Yang, B., Najafi, M., 2021. Targeting of cancer cell death mechanisms by curcumin: Implications to cancer therapy. Basic Clin. Pharmacol. Toxicol. 129, 397–415. https://doi.org/10.1111/BCPT.13648 Zareian, S., Zargar, S.J., Safarian, S., Mozdoori, N., 2022. Investigation of Photodynamic Therapy on Breast Cancer Cell Lines Using LaF3:Tb Nanoparticles Conjugated with Meso-tetra(4-carboxyphenyl) Porphine. J. Clust. Sci. 33, 215–225. https://doi.org/10.1007/S10876-020-01951-Z Zha, Q.B., Zhang, X.Y., Lin, Q.R., Xu, L.H., Zhao, G.X., Pan, H., Zhou, D., Ouyang, D.Y., Liu, Z.H., He, X.H., 2015. Cucurbitacin E Induces Autophagy via Downregulating mTORC1 Signaling and Upregulating AMPK Activity. PLoS One 10, e0124355. https://doi.org/10.1371/JOURNAL.PONE.0124355 Additional Declarations No competing interests reported. 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-2920606\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":199708651,\"identity\":\"39a7000b-42a9-4a40-aece-d69590e81e2a\",\"order_by\":0,\"name\":\"Elham Zeinali\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Tehran\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Elham\",\"middleName\":\"\",\"lastName\":\"Zeinali\",\"suffix\":\"\"},{\"id\":199708652,\"identity\":\"f420176e-2463-4210-920e-b416f2b4b0ac\",\"order_by\":1,\"name\":\"Seyed Jalal Zargar\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYPACGyhtQLyWNAYeUrUchmohBsi3n3384uee84n7GZgffmAouEdYC2NPupllz7PbiT0MbMYSDAbFhLUwM6SxGfAcAGlhMAP6JYGwFjb+Z2yGfw6cA2ph/0acFh6JNObHPAcOALXwEGmLhMQzNmaZA8nGPYd5iiUSiNEi35/G/PHNATvZ9vb2jR8+/CFCCxCwSYApZiAmTgNQ7QciFY6CUTAKRsFIBQAZGDDJDUIBzAAAAABJRU5ErkJggg==\",\"orcid\":\"\",\"institution\":\"University of Tehran\",\"correspondingAuthor\":true,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Seyed\",\"middleName\":\"Jalal\",\"lastName\":\"Zargar\",\"suffix\":\"\"},{\"id\":199708653,\"identity\":\"f1269a69-f310-4015-b731-2fc328d44496\",\"order_by\":2,\"name\":\"Najmeh Mozdoori\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shiraz university, Estahban Higher Education Center\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Najmeh\",\"middleName\":\"\",\"lastName\":\"Mozdoori\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2023-05-11 14:29:26\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-2920606/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-2920606/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":37090127,\"identity\":\"11bce38c-8a80-4aa7-9796-1041e4cab603\",\"added_by\":\"auto\",\"created_at\":\"2023-05-16 14:25:36\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":283505,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCytotoxic effect of Cucurbitacin D on different cell lines tested. A: MCF-7; C: MDA-MB-468; B and D: IC50 value of Cucurbitacin D on MCF-7 and MDA-MB-468 cell lines (*: P ≤ 0.05; **: P ≤ 0.01).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2920606/v1/ec746b0c8a1e18e01b6ab376.jpg\"},{\"id\":37090126,\"identity\":\"ca454feb-3606-4cc3-bf1d-bd86d17d3062\",\"added_by\":\"auto\",\"created_at\":\"2023-05-16 14:25:36\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":183572,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eExpression of apoptotic genes Bax, Bcl-2, caspase 3, and p53 upon treatment with Cucurbitacin D. A: MCF-7; and B: MDA-MB-468. Cells were treated with different concentrations of Cucurbitacin D for 24 hours. Data are shown as fold changes relative to GAPDH expression. n = 3, (*:P ≤ 0.05; **: P ≤ 0.01; ***: P≤0.001; ****: P≤ 0.0001).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2920606/v1/2c19eacb5501c64fdd9d2fe3.jpg\"},{\"id\":37091168,\"identity\":\"c68962bb-9e0c-4538-87df-2082632e7aab\",\"added_by\":\"auto\",\"created_at\":\"2023-05-16 14:33:36\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":154179,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eExpression of autophagic genes atg5 and beclin1 upon treatment with Cucurbitacin D. A: MCF-7; and B: MDA-MB-468. Cells were treated with different concentrations of Cucurbitacin D for 24 hours. Data are shown as fold changes relative to GAPDH expression. n = 3, (*:P ≤ 0.05; **: P ≤ 0.01; ***: P≤0.001; ****: P≤ 0.0001).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2920606/v1/e832e6e0c308d2af80f422b2.jpg\"},{\"id\":37090125,\"identity\":\"7968e785-f34a-4987-b47c-1ff72fa2c27f\",\"added_by\":\"auto\",\"created_at\":\"2023-05-16 14:25:36\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":163539,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eExpression of Akt and PTEN genes upon treatment with Cucurbitacin D. A: MCF-7; and B: MDA-MB-468. Cells were treated with different concentrations of Cucurbitacin D for 24 hours. Data are shown as fold changes relative to GAPDH expression. n = 3, (*:P ≤ 0.05; **: P ≤ 0.01; ***: P≤0.001; ****: P≤ 0.0001).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2920606/v1/f324c7ae77365b897f7f13d7.jpg\"},{\"id\":37208143,\"identity\":\"795ec133-2268-4e5a-890b-88354b2dda38\",\"added_by\":\"auto\",\"created_at\":\"2023-05-18 18:44:25\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":562569,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2920606/v1/df28780e-a621-4816-ae52-1bc5cb843c53.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Purified Cucurbitacin D leads to alterations of apoptotic and autophagic genes expression in MDA-MB-468 and MCF-7 human breast cancer cells\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eBreast cancer is the second most frequent malignancy worldwide after lung cancer and the fifth most common cause of cancer death among women (Aggarwal et al., 2020; Rafatian et al., 2021). Several factors including hormone or estrogen-responsiveness and expression of estrogen or progesterone receptors can determine prognosis of human breast cancer (Shokrollahi Barough et al., 2015). Breast cancer cell lines are extensively used for studying various breast cancers pathogenesis and developing new treatment strategies (Subik et al., 2010; Zareian et al., 2022). Breast cancer cell lines are classified according to their differences in terms of estrogen receptor (ER), progesterone receptor (PR) ERBB2, and HER2/neu expression panel. Estrogen independent or ER negative cell lines such as MDA-MB-468 isolated from high metastatic ER/PR and HER2-negative adenocarcinoma are more aggressive and higher metastatic. However, ER positive cell lines such as MCF-7 isolated from ER/PR positive and HER2-negative adenocarcinoma are less aggressive (Shokrollahi Barough et al., 2015).\\u003c/p\\u003e \\u003cp\\u003eCompared to chemically synthesized cancer medications, plant-derived drugs used in cancer therapy are natural products with less side effects and toxicity (Lee et al., 2010). Cucurbitacins refer to a cluster of tetracyclic triterpenoids isolated from different plant families, including \\u003cem\\u003eCucurbitaceae (Ecballium elaterium)\\u003c/em\\u003e and \\u003cem\\u003eCruciferae\\u003c/em\\u003e. Cucurbitacins are known to have multiple therapeutic effects such as anti-proliferation and anti-cancer (Lee et al., 2010). Cucurbitacins consist of 17 main molecules from cucurbitacin A to cucurbitacin T, among which cucurbitacin B, D, E, I, have been known mainly due to their significant anticancer and antitumor activities (Sikander et al., 2016). Different investigations on Cucurbitacin D (Cu D) revealed its remarkable anticancer activity in many human cancer cell lines (Lee et al., 2010). It was recently reported that Cucurbitacin D is able to induce apoptosis in cancerous cells through different signaling pathways (Kim et al., 2020) .\\u003c/p\\u003e \\u003cp\\u003eApoptosis or programmed cell death is a fundamental event that occurs in a tightly regulated and precise manner and is particularly significant during tissue development and maturation and numerous pathological conditions (Ferhi et al., 2019; Singh et al., 2019). The Bcl-2 family, a group of about 25 genes governing mitochondrial membrane permeability with a pivotal role in the regulation of apoptosis, can be either pro-apoptotic or anti-apoptotic. These proteins have special significance since they can determine if the cell commits to apoptosis or not (Elmore, 2007). The regulation of Bcl-2 family at the transcriptional level seems to be a critical factor in the regulation of cell death (Burlacu, 2003). Bcl-2 is one of the most prominent members of anti-apoptotic proteins which are able to promote cell survival. \\u003cem\\u003eBax\\u003c/em\\u003e, a member of pro-apoptotic proteins, and \\u003cem\\u003ecaspase-3\\u003c/em\\u003e, a key component of a family of proteases with prominent role in apoptotic response, are also among main regulators of apoptosis (Pe\\u0026ntilde;a-Blanco and Garc\\u0026iacute;a-S\\u0026aacute;ez, 2018). DNA damage caused by cancer chemotherapy drugs in some cells, can lead to apoptotic death through a \\u003cem\\u003ep53\\u003c/em\\u003e-dependent pathway. In fact, the tumor suppressor protein p53 has a critical role in \\u003cem\\u003ep53\\u003c/em\\u003e-mediated apoptosis through regulation of the Bcl-2 family members such as Bcl-2 and Bax (Elmore, 2007) .\\u003c/p\\u003e \\u003cp\\u003eAutophagy is one of the key evolutionary mechanisms mediating stress-induced metabolic adaptation and damage control and is implicated in various diseases such as cancer (Codogno and Meijer, 2005; Kroemer et al., 2010). The genes that are responsible for the regulation of autophagy are known as autophagy genes or Atgs. \\u003cem\\u003eAtg5\\u003c/em\\u003e is an autophagic gene which promotes autophagy and may also be involved in pro-apoptotic pathway (Yousefi and Simon, 2007). Beclin1 is the mammalian ortholog of yeast Atg6 which has a critical role in autophagosome formation. Loss of Beclin1 is tightly associated with defects in autophagy (Menon and Dhamija, 2018) .\\u003c/p\\u003e \\u003cp\\u003ePhosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a dual lipid and protein phosphatase whose major substrate is phosphatidylinositol-3,4,5-triphosphate (PIP3), and downstream of which lies the PTEN/PI3K/Akt pathway (Kurose et al., 2001). Therefore, AKT is a serine\\u0026ndash;threonine kinase downstream of PTEN/PI3K, whose activity is regulated by PI3K and its activation results in the suppression of apoptosis. In addition, hyper-activated AKT can promote cell proliferation and cell growth. PTEN/PI3K/Akt pathway members aberrations are common in breast cancer and have been shown to play a distinct role in the pathogenesis of different types of breast tumors (Carnero et al., 2008). Notwithstanding, targeting this pathway will provide an effective therapeutic approach in breast cancer (Li et al., 2017).\\u003c/p\\u003e \\u003cp\\u003eThe anti-cancer effects of Cucurbitacin D on some tumors and tumor-derived cell lines have been proved. However, the anticancer effects of Cucurbitacin D on breast cancer awaits further delineation. The aim of the present study was to examine the effects of Cucurbitacin D on apoptosis as well as the expression levels of \\u003cem\\u003eBc-l2\\u003c/em\\u003e, \\u003cem\\u003eBax\\u003c/em\\u003e, \\u003cem\\u003ecaspase-3\\u003c/em\\u003e, \\u003cem\\u003ep53, Atg5\\u003c/em\\u003e, \\u003cem\\u003eBeclin-1\\u003c/em\\u003e, \\u003cem\\u003ePTEN\\u003c/em\\u003e and \\u003cem\\u003eAkt\\u003c/em\\u003e genes in the MCF-7 and MDA-MB-468 cell lines.\\u003c/p\\u003e\"},{\"header\":\"2. Material and Methods\",\"content\":\"\\u003ch3\\u003e2. 1. Cell Lines, Cell Culture and Treatments\\u003c/h3\\u003e\\n\\u003cp\\u003eTwo human breast cancer cell lines, MCF-7 and MDA-MB-468, were purchased from Iranian Biological Resource center (Tehran, Iran). Cell lines were cultured in DMEM/F12 medium.\\u003c/p\\u003e\\n\\u003ch3\\u003e2. 2. Cell Viability Assay (Cytotoxicity)\\u003c/h3\\u003e\\n\\u003cp\\u003eAnti-proliferative effects of the Cucurbitacin D against MCF-7 and MDA-MB-468 human breast cancer cell lines and cell viability were investigated using the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), assay. Various concentrations of Cucurbitacin D in DMEM/F12 were provided (0.1\\u0026ndash;100 \\u0026micro;M for MCF-7 and 1\\u0026ndash;70 \\u0026micro;M for MDA-MB-468) and cells were treated for an additional overnight incubation. At the end, the absorbance of it was measured at 570 nm using an ELISA plate reader.\\u003c/p\\u003e\\n\\u003ch3\\u003e2. 3. RNA Extraction\\u003c/h3\\u003e\\n\\u003cp\\u003eTotal cell RNA from breast cancer cell lines was extracted using Qiagen RNeasy Mini Kit (QIAGEN, Germany), according to the manufacturer\\u0026rsquo;s protocol.\\u003c/p\\u003e\\n\\u003ch3\\u003e2. 4. Reverse Transcription and Quantitative Real-Time PCR (RT-qPCR)\\u003c/h3\\u003e\\n\\u003cp\\u003eFor cDNA synthesis, 2 \\u0026micro;g of isolated RNA samples were reverse-transcribed with random primer according to the manufacturer\\u0026rsquo;s instructions by Easy cDNA Synthesis Kit (Cat. No. A101161, Pars Tous Biotechnology, Iran). The expression levels of the genes were analyzed based on the cycle threshold (Ct) and relative expression levels were determined as 2\\u003csup\\u003e\\u0026minus;\\u0026Delta;\\u0026Delta;C(t)\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003ch3\\u003e2. 5. Statistical analysis\\u003c/h3\\u003e\\n\\u003cp\\u003eStatistical analyses were carried out using GraphPad Prism 8. Comparison between groups was analyzed by Student\\u0026apos;s independent-samples \\u003cem\\u003et\\u003c/em\\u003e-test. Data are presented as the mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD of three independent experiments and p values\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 were considered statistically significant. (*: P\\u0026thinsp;\\u0026le;\\u0026thinsp;0.05; **: P\\u0026thinsp;\\u0026le;\\u0026thinsp;0.01; ***: P\\u0026thinsp;\\u0026le;\\u0026thinsp;0.001; ****: P\\u0026thinsp;\\u0026le;\\u0026thinsp;0.0001).\\u003c/p\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003ch2\\u003e3. 1. Antiproliferative activities of Cucurbitacin D\\u003c/h2\\u003e\\n\\u003cp\\u003eBased on the articles which we took into consideration in the first place, we decided to create a serial dilution of the drug which was as low as 0.1 \\u0026micro;M to as high as 100 \\u0026micro;M, in MCF-7 and from 1 \\u0026micro;M to 70 \\u0026micro;M, in MDA-MB-468. According to the graph which was accomplished by MTT results inserted in the excel program, IC50 was calculated based on the equations of each line. In the absence of cucurbitacin D, MCF-7 cells show more than 98% viability. Upon addition of cucurbitacin D, the MCF-7 cell viability was reduced to 88%, at 0.1 \\u0026micro;M of cucurbitacin D until it reaches 83% at 0.5 \\u0026micro;M. Then the cell viability experiences a slight increase followed by a gradually drop till reaches 68% at 20 \\u0026micro;M. There was a significant decrease in the cell viability after cucurbitacin D exposure, especially at high concentrations (\\u0026ge;\\u0026thinsp;50 \\u0026micro;g/ml). (mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD, n\\u0026thinsp;=\\u0026thinsp;3). The same behavior was seen in MDA-MB-468 cell lines. The cell viability of MDA-MB-468 cells stands in about 99% in absence of Cucurbitacin D and subsequently decreases from 80% at 1 \\u0026micro;M to 70% at 20 \\u0026micro;M. A sudden drop is shown in cell viability at concentrations higher than 20 \\u0026micro;M. The half-maximal concentrations of proliferation inhibition or IC50 which reduced cell viability by 50% after 24 hours of incubation were 30 and 25 \\u0026micro;M for MCF-7 and MDA-MB-468 cells, respectively. These IC50s as well as the concentrations in which the percentages of viability were 30% and 80% were chosen for subsequent experiments (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e\\n\\u003cp\\u003eThese concentrations are 50 and 0.5 \\u0026micro;M of Cu D for MCF-7 and 50 and 1 \\u0026micro;M for MDA-MB 468 cell line.\\u003c/p\\u003e\\n\\u003ch2\\u003e3. 2. Effect of Cucurbitacin D on the mRNA expression profile of apoptosis related genes\\u003c/h2\\u003e\\n\\u003cp\\u003eThe expression levels of four key apoptotic genes were evaluated in MCF-7 and MDA-MB-468 cells cultured in the presence of Cucurbitacin D for 24 hours. The expression levels of \\u003cem\\u003eBax\\u003c/em\\u003e, \\u003cem\\u003eBcl-2\\u003c/em\\u003e, \\u003cem\\u003ecaspase3\\u003c/em\\u003e, and \\u003cem\\u003ep53\\u003c/em\\u003e in MCF-7 and MDA-MB-468\\u003cstrong\\u003e \\u003c/strong\\u003ecell lines, treated with various concentrations of Cucurbitacin D can be seen in Figure 2.\\u003c/p\\u003e\\n\\u003cp\\u003eTreated MCF-7 cells exhibited a significant increased expression levels of \\u003cem\\u003eBcl-2\\u003c/em\\u003e at all three Cucurbitacin D concentrations. However, no significant difference was shown in treated MCF-7 cells regarding expression profiles of two apoptotic genes, \\u003cem\\u003eBax\\u003c/em\\u003e and \\u003cem\\u003ep53\\u003c/em\\u003e, at 30 \\u0026mu;M Cucurbitacin D (\\u003cem\\u003eP \\u003c/em\\u003e= 0.343 and 0.174, respectively). Interestingly, these genes, \\u003cem\\u003eBax\\u003c/em\\u003e and \\u003cem\\u003ep53\\u003c/em\\u003e, showed significant decreased expression levels at 0.5 and 50\\u0026mu;M Cucurbitacin D (Figure 2A).\\u003c/p\\u003e\\n\\u003cp\\u003eMDA-MB-468 exhibited similar trend to MCF-7 cells regarding the expression levels of all four apoptotic genes so that in these cells\\u0026rsquo; expression levels of \\u003cem\\u003eBcl-2\\u003c/em\\u003e had a significant increase\\u003cem\\u003e \\u003c/em\\u003eat all three concentrations of Cucurbitacin D compared with untreated control cells. \\u003cem\\u003eBax\\u003c/em\\u003e and \\u003cem\\u003ep53 \\u003c/em\\u003egenes had significant decreased expression levels at 1 and 50 \\u0026mu;M Cucurbitacin and they failed to show any significant difference between treated and control cells at 25 \\u0026mu;M (Figure 2B). No significant changes were observed in the expression levels of \\u003cem\\u003ecaspase-3\\u003c/em\\u003e at all tested Cucurbitacin D concentrations in both cell lines.\\u003c/p\\u003e\\n\\u003ch2\\u003e3. 3. Effects of Cucurbitacin D on the mRNA expression profile of autophagic genes \\u003c/h2\\u003e\\n\\u003cp\\u003eIn the next step, the expression profiles of \\u003cem\\u003eAtg5\\u003c/em\\u003e and \\u003cem\\u003eBeclin1\\u003c/em\\u003e were measured as two important autophagic genes. In MCF-7 cell line, an increase in the concentration of Cucurbitacin D results in the down-regulation of both \\u003cem\\u003eAtg5\\u003c/em\\u003e and \\u003cem\\u003eBeclin1\\u003c/em\\u003e genes so that their expression was enhanced at low Cucurbitacin D concentrations (\\u003cem\\u003eP \\u003c/em\\u003e= 0.01 and \\u0026gt;0.0001, respectively) while increased concentrations (50\\u0026mu;M) led to a reduction in their expressions (\\u003cem\\u003eP \\u003c/em\\u003e= 0.0004 and 0.001, respectively). \\u003cem\\u003eAtg5\\u003c/em\\u003e and \\u003cem\\u003eBeclin1\\u003c/em\\u003e genes levels did not change remarkably at IC50 (\\u003cem\\u003eP \\u003c/em\\u003e= 0.16 and 0.76, respectively). Nevertheless, in MDA-MB-468 cell line autophagic genes indicated a different expression pattern and \\u003cem\\u003eBeclin1\\u003c/em\\u003e expression showed similar pattern to \\u003cem\\u003eAtg5\\u003c/em\\u003e. In this cell line, the expression levels of the autophagic genes\\u003cem\\u003e \\u003c/em\\u003eshowed significant upregulation only at 25 \\u0026mu;M and their levels did not change at concentrations other than IC50 (Figure 3).\\u003c/p\\u003e\\n\\u003ch2\\u003e3. 4. Effect of Cucurbitacin D on the mRNA expression profile of cell survival genes\\u003c/h2\\u003e\\n\\u003cp\\u003eA preliminary comparison between the relative mRNA expressions of \\u003cem\\u003eAkt\\u003c/em\\u003e and \\u003cem\\u003ePTEN\\u003c/em\\u003e in treated MCF-7 cells and control cells revealed that enhanced Cucurbitacin D concentration (50\\u0026mu;M) led to a significant reduction in expression levels of \\u003cem\\u003eAkt\\u003c/em\\u003e and increase in \\u003cem\\u003ePTEN\\u003c/em\\u003e levels. Both MCF-7 and MDA-MB-468 cell lines demonstrated significant increased expression of \\u003cem\\u003ePTEN\\u003c/em\\u003e at different concentrations of Cucurbitacin D (Figure 4). MCF-7 and MDA-MB-468 breast cancer cells showed significant lower expression levels of \\u003cem\\u003eAkt\\u003c/em\\u003e only at enhanced Cucurbitacin D concentrations (50\\u0026mu;M).\\u003c/p\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003eUnfortunately, despite improved cure rate, there is still limited success in breast cancer treatment so that more than 400,000 patients are estimated to die from this malignancy annually (Li et al., 2016). Natural therapies based on medicinal plants or traditional herbal remedies have gained increasing attention for the cancer prevention (Jafargholizadeh et al., 2018). Cucurbitacins belonging to a group of tetracyclic triterpenoids with a wide range of biological effects, are able to prevent cell growth and induce apoptosis in a wide variety of cancer cell lines (Kim et al., 2020). Since cucurbitacins were identified with emerging anti-cancer activities, such as pro-apoptosis and autophagy induction, they have been reported to be a potential candidate for various cancer therapies, including gastric, breast, ovarian, and lung (Luo et al., 2019). The underlying mechanisms of Cucurbitacin D anticancer effects in human breast cancer are still elusive. In this study, the effect of different concentrations of Cucurbitacin D on cell viability and expression levels of some genes related to the apoptosis, autophagy and cell survival was investigated in MCF-7 and MDA-MB-468 human breast cancer cell lines. MDA-MB-468 was used as an estrogen independent or ER-negative cell line which is triple negative and more aggressive and metastatic than MCF-7 utilized as an estrogen dependent or ER-positive cell line.\\u003c/p\\u003e \\u003cp\\u003eResults of cell survival assay after 24 h of MCF-7 and MDA-MB-468 cells exposure to the Cucurbitacin D revealed that it can significantly reduce cell survival in a dose-dependent manner. The results confirmed the cytotoxic effect of Cucurbitacin D on the two breast cancer cell lines with IC50 values of 30 \\u0026micro;M in MCF-7 and 25 \\u0026micro;M in MDA-MB-468 cells. This is in conjunction with the results of a study by Kim et al. that showed Cucurbitacin D inhibits cell proliferation and induces G2/M phase cell cycle arrest in MDA-MB-231 cells (Kim et al., 2013). Further studies with cucurbitacin D showed that it is able to inhibit proliferation and induce apoptosis in doxorubicin-resistant breast cancer MCF-7/ADR cells (Luo et al., 2019). Besides, Kim et al. reported that Cucurbitacin D, isolated from \\u003cem\\u003eTrichosanthes kirilowii\\u003c/em\\u003e (TKE), can induce apoptosis through activation of caspases and JNK in hepatocellular carcinoma cells (Kim et al., 2013). In fact, cucurbitacins can exert their anticancer effects through various targets (Luo et al., 2019). JAK-STAT, AKT-PKB, and MAPK pathways are some of the most common pathways involved in cancer cells, all of which are targets of the cucurbitacin family (Lee et al., 2010). Cucurbitacins can act as STAT3 inhibitors in cancer cells. JAK-STAT pathway inhibition would affect different downstream targets involved in cell growth and apoptosis. The anticancer mechanism by which cucurbitacins act in breast cancer cells is still not clear. An investigation by Tannin-Spitz et al. who exposed two breast cancer cell lines, MCF-7 and MDA-MB-231, to cucurbitacin B/E showed that these cucurbitacins can increase the STAT3 phosphorylation (Lee et al., 2010). In addition, cucurbitacin mediates apoptosis through mitochondrial-related pathway, which can be characterized by the Bcl-2 down-regulation and Bax up-regulation, that eventually leads to the caspase activation (Luo et al., 2019).The next step in this investigation was to examine whether Cucurbitacin D exert its cytotoxic effects on breast cancer cell lines through apoptotic genes or not.\\u003c/p\\u003e \\u003cp\\u003eThe Bcl-2 family proteins have special significance since they can determine if the cell commits to apoptosis or aborts the process. The main mechanism through which the Bcl-2 family of proteins act is the regulation of cytochrome \\u003cem\\u003ec\\u003c/em\\u003e release via alteration of mitochondrial membrane permeability. Bcl-2 is one of the most prominent members of anti-apoptotic proteins (Elmore, 2007). Strikingly, both breast cancer cell lines used in the current study exhibited a significant increased expression levels of \\u003cem\\u003eBcl2\\u003c/em\\u003e at all Cucurbitacin D concentrations.\\u003c/p\\u003e \\u003cp\\u003eBax is a well-known pro-apoptotic molecule with about 21% amino acid identity with Bcl-2 (Burlacu, 2003). Interestingly, in this investigation \\u003cem\\u003eBax\\u003c/em\\u003e showed significant decreased expression levels at both Cucurbitacin D concentrations higher or lower than the IC50 in MCF-7 and MDA-MB-468 breast cancer cells. Previous studies revealed that cells with high Bax/Bcl-2 ratio are more susceptible to the apoptosis due to the activation of caspases 3, 8 and 9 (Lee et al., 2012). Fascinatingly, this ratio was less than one in all the Cucurbitacin D treatments in our study. It can be concluded that none of the breast cancer cell lines treated with Cucurbitacin D are prone to apoptosis and apoptosis is not stimulated by Cucurbitacin D in these cancerous cells at least through changes in the expression levels of \\u003cem\\u003eBcl-2\\u003c/em\\u003e and \\u003cem\\u003eBax\\u003c/em\\u003e.\\u003c/p\\u003e \\u003cp\\u003eThe tumor suppressor protein p53 has a critical role in the regulation of the Bcl-2 family. Additionally, irradiation or drugs used for cancer chemotherapy results in DNA damage in some cells, which can lead to apoptotic death through a \\u003cem\\u003ep53\\u003c/em\\u003e-dependent pathway. Importantly, Bcl-2 and Bax expression is shown to be regulated by the \\u003cem\\u003ep53\\u003c/em\\u003e tumor suppressor gene (Elmore, 2007). Notably, our data indicate that the expression level of \\u003cem\\u003ep53\\u003c/em\\u003e did not change at IC50 concentrations, while its expression decreased significantly at Cucurbitacin D concentrations other than IC50 in the two tested cancerous cell lines. This observation can confirm the results of \\u003cem\\u003eBcl2\\u003c/em\\u003e and \\u003cem\\u003eBax\\u003c/em\\u003e expression showing that apoptosis is not triggered in the breast cancer cells treated with Cucurbitacin D through these two genes. Moreover, \\u003cem\\u003eBcl-2\\u003c/em\\u003e and \\u003cem\\u003eBax\\u003c/em\\u003e expression dysregulation can be due to the deregulated expression levels of \\u003cem\\u003ep53\\u003c/em\\u003e. A study by Yuan et al (Lee et al., 2012). using GBM cells treated with cucurbitacin I for 48 h confirmed significant increased Bax and cleaved caspase-3 but decreased antiapoptotic proteins such as Bcl-2 and Bcl-xL in a dose-dependent manner (Jafargholizadeh et al., 2018). Further, Kim et al. reported that Cucurbitacin D induces apoptosis via activation of caspases in hepatocellular carcinoma cells (Kim et al., 2013).\\u003c/p\\u003e \\u003cp\\u003eCaspase-3 is considered to be the most important executioner caspase whose activity lead to the apoptosis (Elmore, 2007). Noticeably, MCF-7 and MDA-MB-468 cells showed no significant changes in the expression levels of \\u003cem\\u003ecaspase-3\\u003c/em\\u003e at all tested Cucurbitacin D concentrations. Analysis of the expression levels of four important apoptotic genes, including \\u003cem\\u003eBCl-2\\u003c/em\\u003e, \\u003cem\\u003eBax\\u003c/em\\u003e, \\u003cem\\u003ep53\\u003c/em\\u003e and \\u003cem\\u003ecaspase-3\\u003c/em\\u003e and considering the role of Cucurbitacin D in apoptosis indicate that Cucurbitacin D exerts its cytotoxic effect on breast cancer cells through pathways other than the caspase-dependent cell death. So, the most important cell death mechanism in these treatments is not canonical apoptosis. One possibility could be that Cucurbitacin D may affect breast cancer cells viability through triggering non-canonical apoptosis which is independent of caspases. In other words, Cucurbitacin D could limit caspase-dependent apoptosis while it induces non-canonical apoptosis (Jafargholizadeh et al., 2018). Another possibility is that Cucurbitacin D might induce apoptosis mainly through the dysregulation of other members of the Bcl-2 family. It goes without saying that further investigations are required to throw further light on the effects of Cucurbitacin D on breast cancer cells apoptosis and its related genes.\\u003c/p\\u003e \\u003cp\\u003eDespite of the fact that autophagy is a survival mechanism, some changes in autophagic activity can lead to the cell death. Autophagy has been reported to have a key role in tumor suppression so that reduced autophagic activity can be found in some cancers and sufficient autophagy is vital for tumor suppression (Yousefi and Simon, 2007). It has been documented that Cu E, Cucurbitacin D, and Cu I are capable of inducing autophagy in different cancer cell lines (Niu et al., 2016). It was previously reported that Cu E and Cu I-induced autophagy is dependent on ATG5 and Beclin 1 expression (Zha et al., 2015).\\u003c/p\\u003e \\u003cp\\u003eBeclin1 is necessary for autophagic pathway. The functional and structural interaction between Beclin1 and Bcl-2 is a key event in the considerable crosstalk between autophagy and apoptosis and the regulation of autophagy (Codogno and Meijer, 2005; Nikoletopoulou et al., 2013). The p53 deacetylation can induce autophagy by reducing the interaction of Beclin-1 and Bcl-2. \\u003cem\\u003eAtg5\\u003c/em\\u003e is another well-known autophagic gene which is critical for the formation of autophagosomes and regulation of autophagic cell death. Noteworthy, caspase-mediated cleavage of Beclin1 and ATG5 can switch autophagy to apoptosis (Nikoletopoulou et al., 2013). Our results show that in MCF-7 cell line, an increase in the concentration of Cucurbitacin D results in the down-regulation of both \\u003cem\\u003eBeclin1\\u003c/em\\u003e and \\u003cem\\u003eAtg5\\u003c/em\\u003e autophagic genes expression levels. So, it can be suggested that at Cucurbitacin D concentrations lower than IC50 (30 \\u0026micro;M), MCF-7 cells are more prone to autophagy, while Cucurbitacin D concentration of 50 \\u0026micro;M does not exert cytotoxic effects on MCF-7 cells through autophagy. Remarkably, these two autophagic genes expression analysis indicated that the mechanism through which Cucurbitacin D acts in MDA-MB-468 breast cancer cells is completely different from that in the MCF-7 cells. MDA-MB-468 cells showed significant increased expression levels of both \\u003cem\\u003eBeclin1\\u003c/em\\u003e and \\u003cem\\u003eAtg5\\u003c/em\\u003e only at concentration of 25 \\u0026micro;M (IC50). The expression levels of these genes did not change significantly at Cucurbitacin D concentrations less or higher than IC50 in these cells. It can be concluded that autophagy is triggered in MDA-MB-468 at IC50 of Cucurbitacin D.\\u003c/p\\u003e \\u003cp\\u003eTo further investigate the mechanism by which Cucurbitacin D affects breast cancer cells viability, in the next step we evaluated the expression levels of two prominent cell survival related genes. PTEN/PI3K/Akt pathway plays a vital role in breast carcinogenesis so that its aberrations are common in this cancer. The PTEN/PI3K/Akt pathway mutational aberrations are markedly different among the different breast cancer subtypes and are most common in hormone receptor\\u0026ndash;positive breast tumors. PTEN loss was reported to be associated with adverse outcomes in breast cancer (Stemke-Hale et al., 2008). PTEN has also been reported to be directly associated with p53 through increasing protein levels, stability and transcriptional activity of p53 (Carnero et al., 2008). In addition, previous investigations showed that PTEN activation might act as an upstream molecule of autophagy so that its loss of function can cause a strong inhibition of the formation and maturation of autophagosomes (Aquila et al., 2020). Ectopic expression of PTEN results in cell death in the glioma and breast cancer cell lines (Kurose et al., 2001). Noticeably, both MCF-7 and MDA-MB-468 breast cancer cell lines exhibited a significant increased expression levels of \\u003cem\\u003ePTEN\\u003c/em\\u003e after 24h treatment with different Cucurbitacin D concentrations. A recent study by Niu. et al revealed that Cu B inhibited SH-SY5Y cells proliferation through upregulation of PTEN (Niu et al., 2016).\\u003c/p\\u003e \\u003cp\\u003ePTEN indirectly inhibits Akt phosphorylation which results in decreased Akt activity, the kinase downstream of PTEN/PI3K/Akt (Carnero et al., 2008). This kinase plays an essential role in the PTEN/PI3K/Akt pathway. Loss of PTEN function can lead to the constitutive activation of Akt (Sos et al., 2009). The activation of serine\\u0026ndash;threonine kinase Akt stimulates cell cycle progression and cell survival in a wide variety of cells, including cancer cells (Carnero et al., 2008). AKT-targeted therapies are being introduced into trials (Stemke-Hale et al., 2008). Noteworthy, our observations showed that \\u003cem\\u003eAkt\\u003c/em\\u003e expression level decreased in both breast cancer cell lines after treatment with all concentrations of Cucurbitacin D. However, this decrease was only significant at the concentrations higher than IC50. PTEN suppress breast cancer growth and can cause cell cycle arrest at G1 (Paramio et al., 1999; Yu et al., 2021).\\u003c/p\\u003e \\u003cp\\u003eCytotoxic effects of Cucurbitacin D on breast cancer cells may involve some mechanisms including the PARP cleavage, MAPK pathway, decreasing pSTAT3 and JAK3 levels, expression of active caspase-3, and decreasing STAT3 downstream targets, even up-regulation of PTEN (Aquila et al., 2020) which needs to be further investigated. According to the results of the current investigation, it seems that Cucurbitacin D affects different breast cancer cells through different mechanisms at different concentrations so that it may induce autophagy at a particular concentration in a particular cell line while it induces apoptosis at other concentrations in another one (Jafargholizadeh et al., 2018). Besides, differences in the gene\\u0026rsquo;s expression levels under the Cucurbitacin D treatment, observed in MCF-7 and MDA-MB-468 cell lines can be mainly due to the origin, genetic and macromolecular structure differences between these cell lines. Therefore, as it was previously pointed out by Raikhlin-Eisenkraft et al., the toxicity of cucurbitacins can be affected by many factors. The discrepancy between our results and previous studies can be partly explained by the extent of purity as well as the type of solvents used for elution and dilution of Cucurbitacin D (Lee et al., 2010). According to the studies performed by Naoki Wakimoto et al there was no significant connection between the sensitivity to growth inhibition by Cucurbitacin B and molecular characteristics such as ER, Her-2/neo or p53 status. This might be an effective treatment against triple negative breast cancer (Wakimoto et al., 2008). Furthermore, Tehila Tannin-Spitz and et al reported that cucurbitacin glucosides isolated from \\u003cem\\u003eCitrullus colocynthis\\u003c/em\\u003e can have anti-proliferative activity on both estrogen-dependent and estrogen-independent human breast cancer cells by causing G2/M phase arrest. It is reasonable to hypothesize that cucurbitacin glucosides can be useful for chemotherapy of both estrogen-dependent and independent breast cancers (Tannin-Spitz et al., 2007). However, the differences between the gene\\u0026rsquo;s aberrations in our breast cancer cell lines propose that these genes may have a distinct role in the pathophysiology and therapy responsiveness of various subtypes of breast cancer.\\u003c/p\\u003e\"},{\"header\":\"5. Conclusion\",\"content\":\"\\u003cp\\u003eThis study provides molecular insight into the effects of Cucurbitacin D on breast cancer. We examined the effects of different concentrations of Cucurbitacin D on apoptosis as well as the expression levels of autophagic and cell survival genes in the MCF-7 and MDA-MB-468 breast cancer cell lines. The results of the cell viability assay confirmed the cytotoxic effect of Cucurbitacin D on the two breast cancer cell lines. In both cell lines apoptosis is not induced by Cucurbitacin D through canonical pathway. Non-canonical apoptosis pathways or dysregulation of other the Bcl-2 family members should be evaluated for further studies. Cucurbitacin D also triggers autophagy in MDA-MB-468 and MCF-7 in various concentrations (in IC50 and concentration lower than IC50; respectively). To get a more accurate conclusion, protein assays should be performed for key genes in the autophagy and apoptosis pathways.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthical Approval\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis declaration is \\u0026ldquo;not applicable\\u0026rdquo;, because it is necessary for human and/ or animal studies.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no conflict of interest.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors\\u0026apos; contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eElham Zeinali has worked on the experimental part.\\u003c/p\\u003e\\n\\u003cp\\u003eSeyed Jalal Zargar has supervised this research and he has written it.\\u003c/p\\u003e\\n\\u003cp\\u003eNajmeh Mozdoori has contributed in writing this paper.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis research was funded by Iran National Science Foundation (INSF), Grant No. 90004329. We thank the Research Council of the University of Tehran for their financial support of this research.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe data has been defined in this article and it doesn\\u0026rsquo;t have any supplementary data.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eAggarwal, V., Priyanka, K., Tuli, H.S., 2020. Emergence of Circulating MicroRNAs in Breast Cancer as Diagnostic and Therapeutic Efficacy Biomarkers. Mol. Diagnosis Ther. https://doi.org/10.1007/s40291-020-00447-w\\u003c/li\\u003e\\n\\u003cli\\u003eAquila, S., Santoro, M., Caputo, A., Panno, M.L., Pezzi, V., De Amicis, F., 2020. The Tumor Suppressor PTEN as Molecular Switch Node Regulating Cell Metabolism and Autophagy: Implications in Immune System and Tumor Microenvironment. 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Beclin 1 Phosphorylation - at the Center of Autophagy Regulation. Front. cell Dev. Biol. 6. https://doi.org/10.3389/FCELL.2018.00137\\u003c/li\\u003e\\n\\u003cli\\u003eNikoletopoulou, V., Markaki, M., Palikaras, K., Tavernarakis, N., 2013. Crosstalk between apoptosis, necrosis and autophagy. Biochim. Biophys. Acta 1833, 3448\\u0026ndash;3459. https://doi.org/10.1016/J.BBAMCR.2013.06.001\\u003c/li\\u003e\\n\\u003cli\\u003eNiu, Y., Sun, W., Lu, J.J., Ma, D.L., Leung, C.H., Pei, L., Chen, X., 2016. PTEN activation by DNA damage induces protective autophagy in response to Cucurbitacin B in hepatocellular carcinoma cells. Oxid. Med. Cell. Longev. 2016. https://doi.org/10.1155/2016/4313204\\u003c/li\\u003e\\n\\u003cli\\u003eParamio, J.M., Navarro, M., Segrelles, C., G\\u0026oacute;mez-Casero, E., Jorcano, J.L., 1999. PTEN tumour suppressor is linked to the cell cycle control through the retinoblastoma protein. 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PLoS One 10, e0124355. https://doi.org/10.1371/JOURNAL.PONE.0124355\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"Breast cancer, Cucurbitacin D, Cell viability, Apoptosis, Autophagy\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-2920606/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-2920606/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eBreast cancer is the second most frequent malignancy worldwide. The use of plant-derived drugs in cancer therapy are widely considered in the treatment of different malignancies including breast cancer. Cucurbitacin D (Cu D) is able to induce apoptosis in cancerous cells through different signaling pathways. The aim of this study was to examine the effect of different concentrations of Cucurbitacin D on viability, death pattern as well as the expression alterations of \\u003cem\\u003eBcl-2\\u003c/em\\u003e, \\u003cem\\u003eBax\\u003c/em\\u003e, \\u003cem\\u003ecaspase-3\\u003c/em\\u003e, \\u003cem\\u003ep53, Atg5\\u003c/em\\u003e, \\u003cem\\u003eBeclin-1\\u003c/em\\u003e, \\u003cem\\u003ePTEN\\u003c/em\\u003e and \\u003cem\\u003eAkt\\u003c/em\\u003e genes in the MCF-7 (ER positive) and MDA-MB-468 (triple negative) breast cancer cell lines. Two breast cancer cell lines (MCF-7 and MDA-MB-468) were cultured and treated with different concentrations of the purified Cucurbitacin D. Anti-proliferative effects of the Cucurbitacin D on both breast cancer cell lines viability was investigated using the MTT assay. Real-time PCR was applied to evaluate the expression levels of the genes upon Cucurbitacin D therapy. Significant dose-dependent and anti-proliferative effects of the Cucurbitacin D were observed on MCF-7 and MDA-MB-468 cells after 24 h with IC50 value about 30 and 25 \\u0026micro;M, respectively (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01). Significant changes in expression of the genes in the breast cancer lines (MCF-7 and MDA-MB-468), were observed under different concentrations of Cucurbitacin D. Our results confirmed that the Cucurbitacin D may influence breast cancer cell lines viability at specific doses and by altering the expression of these genes. The differences between the gene\\u0026rsquo;s aberrations in our breast cancer cell lines propose that these genes can have a distinct role in the pathophysiology and therapy responsiveness of various subtypes of breast cancer.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Purified Cucurbitacin D leads to alterations of apoptotic and autophagic genes expression in MDA-MB-468 and MCF-7 human breast cancer cells\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2023-05-16 14:25:31\",\"doi\":\"10.21203/rs.3.rs-2920606/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"12869c36-a432-4329-bb68-510e3d97009d\",\"owner\":[],\"postedDate\":\"May 16th, 2023\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2023-05-18T18:44:13+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2023-05-16 14:25:31\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-2920606\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-2920606\",\"identity\":\"rs-2920606\",\"version\":[\"v1\"]},\"buildId\":\"wLkW0s4AflPzk-lpfg-fK\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}