Thymoquinone inhibits tumor progression and promotes chemo-sensitivity via modulation of P21/PI3K/Akt axis in colorectal cancer cells

Thymoquinone inhibits tumor progression and promotes chemo-sensitivity via modulation of P21/PI3K/Akt axis in colorectal 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Thymoquinone inhibits tumor progression and promotes chemo-sensitivity via modulation of P21/PI3K/Akt axis in colorectal cancer cells Peng Zhang, Shaowen Li, Pei Zhang, Yuanfang Sun This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5941352/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 Natural thymoquinone has been extensively used as a chemopreventive agent and has shown potent anticarcinogenic activity against a broad range of human malignancies. However, the underlying mechanisms and involving signaling pathways are still not well studies. Therefore, this study is aimed to evaluate the effects of thymoquinone on the increasing doxorubicin chemosensitivity via targeting P21 and PI3K/AKT signaling. Caco-2 cells were treated with thymoquinone. MTT assay were applied to assess the impact of different dose of thymoquinone on the doxorubicin cytotoxicity. The mRNA and protein expression levels of PI3K, Akt, P21, P53, Cyc D1, Cdk4, Cdk6, PCNA, Bax, Bcl-2 and caspase-3 were assessed by qRT-PCR and western blotting. A cell death ELISA commercial kits were used to measure apoptosis. We found that thymoquinone treatment significantly decreased proliferation rate in Caco-2 colorectal cancer cells. The survival rate of cells was reduced significantly when doxorubicin was combined with thymoquinone. Thymoquinone upregulated p21, P53 and downregulated Cyc D1, Cdk4, Cdk6, PCNA, as well as suppressed PI3K/Akt signaling pathway. We conclude that thymoquinone induces doxorubicin sensitivity in colorectal cancer cells through targeting p21 and the PI3K/AKT pathway, thus implicating its importance in chemotherapy for colorectal cancer. Cisplatin colorectal cancer thymoquinone siRNA apoptosis p21 P53 Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Colorectal cancer is one of the most prevalent cancers globally, with a significant impact on public health. It is the third most commonly diagnosed cancer in men and the second in women ( 1 ). The incidence rates vary widely across regions, with the highest rates observed in developed countries, while lower rates are found in Africa and South-Central Asia ( 2 ). This variation is largely attributed to differences in dietary habits, lifestyle factors, and the availability of screening programs. Additionally, genetic predispositions, dietary factors, and lifestyle choices such as smoking, alcohol consumption, and physical inactivity contribute to the risk. Public health initiatives focusing on awareness, early detection, and lifestyle modifications are critical in reducing the global burden of colorectal cancer ( 3 ). Early detection and treatment are crucial for improving survival rates, as advanced stages of colorectal cancer can spread to other parts of the body. Regular screening and awareness are essential for early diagnosis and effective management of this condition ( 4 ). Natural compounds have gained attention in the treatment of colorectal cancer due to their potential therapeutic benefits and lower toxicity compared to conventional chemotherapy ( 5 ). Phytochemicals such as flavonoids, polyphenols, and alkaloids from fruits, vegetables, and herbal medicines have shown potential in modulating signaling pathways involved in colorectal cancer progression ( 6 ). These natural compounds offer a promising avenue for developing novel therapeutic strategies, either as standalone treatments or in combination with existing therapies, to improve outcomes for colorectal cancer patients. Thymoquinone, a bioactive compound derived from black cumin seeds (Nigella sativa), has shown significant promise in the treatment of colorectal cancer ( 7 ). It exhibits potent anticancer activities, including the induction of apoptosis, inhibition of cell proliferation, and suppression of metastasis ( 8 ). It has shown significant promise in the treatment of colorectal cancer by modulating various signaling pathways, including the PI3K/AKT pathway ( 9 ). The PI3K/AKT pathway is frequently activated in colorectal cancer and plays a crucial role in promoting cell survival, proliferation, and resistance to apoptosis ( 10 ). Thymoquinone has been found to inhibit the PI3K/AKT pathway, leading to reduced tumor growth and enhanced chemosensitivity ( 11 ). By suppressing AKT phosphorylation, thymoquinone can induce apoptosis and inhibit the proliferation of colorectal cancer cells. This inhibition of the PI3K/AKT pathway also contributes to the suppression of metastasis, further highlighting the therapeutic potential of thymoquinone in colorectal cancer treatment ( 11 ). The ability of thymoquinone to target the PI3K/AKT signaling pathway underscores its potential as a therapeutic agent, either as a standalone treatment or in combination with existing therapies, to improve outcomes for colorectal cancer patients. Further research is needed to fully elucidate the mechanisms by which thymoquinone modulates this pathway and its clinical applications. Therefore, this study is aimed to evaluate the effects of thymoquinone on the increasing doxorubicin chemosensitivity via targeting P21 and PI3K/AKT signaling. 2. Material and methods 2.1.Cell culture and transfection We obtained the Caco-2 cells from the Pastor Institute cell bank for the study of colorectal cancer (Tehran, Iran). RPMI-1640 cell culture medium containing 10% fetal bovine serum (FBS) and 100 U/mL antibiotic solution (penicillin/ streptomycin) was used to maintain and grow the cells. After reaching to exponential growth condition, cells were transfected with Lipofectamine™2000 reagent containing scrambled negative controls (si-NC) and p21 siRNA (si-p21) purchased from BioSciences co. 2.2. Evaluation of cell viability In order to determine the viability of Caco-2 cells, 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) was used as a staining method. 96-well plates were used to culture the cells and then treated with doxorubicin or thymoquinone at different concentrations. Incubation was extended for another 4 hours with fresh medium containing 20 µL MTT solution after 48 hours. To dissolve the formed crystals, 150 µL of DMSO were used. Three replicate experiments were conducted at 490 nm to measure absorbance. 2.3. qRT-PCR For measuring the mRNA expression levels of P21, P53, Cyc D1, Cdk4, Cdk6, PCNA, PI3K, Akt, Bax, Bcl-2, and capsase-3, qRT-PCR was performed. In all groups, total RNA was extracted from cells using a specific RNA isolation kit. RNA samples were analyzed for quality and quantity, and reverse transcription to cDNA was performed according to the manufacturer's instructions. q-PCR was applied by using the SYBR Green and specific primers on Rotor-Gene™ 6000 system (Corbett Life Science, Mortlake, Australia). 2.4. Western blotting Preparation of suitable protein samples for western blotting was performed using RIPA buffer (Sigma-Aldrich). Protein concentration was also determined by the Bradford method, and proteins were separated by 10% SDS-polyacrylamide gel electrophoresis. After this step, PVDF membranes were used to transfer separated proteins. At room temperature, the membrane was then placed in a buffer containing 5% bovine serum albumin for 2 hours, and then polyclonal antibodies for P21, P53, Cyc D1, Cdk4, Cdk6, PCNA, PI3K, Akt, Bax, Bcl-2, capsase-3 and β-actin were added and incubated at 4ºc overnight. Subsequently, secondary antibodies were added and incubated for 2 h at room temperature. The final step was to expose and photograph the slide. 2.5. ELISA cell death assay ELISA cells death assay was applied to measure the apoptosis rate of Caco-2 cells treated with DOX and thymoquinone. All steps were followed in accordance to the manufacturer’s protocol. 2.6. Statistical analysis All data were expressed as mean ± SD. Kolmogorov-Smirnov test was used to assess normality of the data and one-way analysis of variance (ANOVA), followed Tukey’s test were performed for evaluation of multiple comparison using GraphPad prism software. The values were considered statistically significant at P < 0.05. 3. Results 3.1. The effects of thymoquinone on the doxorubicin mediated inhibition of cells viability and proliferation in Caco-2 cells MTT assay was performed to measure the cytotoxic effects of doxorubicin, thymoquinone, and their combination following Caco-2 cells exposure to these treatments. Upon 48-h incubation, cell viability was measured (Fig. 1 ). By increasing the concentration of doxorubicin from 0 to 5 µM and thymoquinone from 0 to 800 µM, its inhibitory effect on cell viability was increased, such that the cells viability was almost completely suppressed in higher concentrations of doxorubicin and also thymoquinone. In Caco-2 cell line, doxorubicin IC50 was 2.4 ± 1 µM and thymoquinone IC50 was 470 ± 12 µM after treating the cells for 48 hours (Fig. 1 a and 1 b). As shown in Fig. 1 c, simultaneous exposure of Caco-2 cells to different amounts of doxorubicin and 470 µM of thymoquinone led to potentiation of doxorubicin mediated inhibition of cell viability, such that the IC50 value of doxorubicin was decreased from 2.4 to 1.2 µM. 3.2. Thymoquinone modulates Caco-2 cells' apoptotic response to doxorubicin Our next step was to determine whether pairing doxorubicin and thymoquinone increased Caco-2 cell apoptosis. When cells were treated with doxorubicin or thymoquinone, the apoptosis rate was significantly higher than when they were not treated. This increase in apoptosis rate can be seen in the data, where the percentage of apoptotic cells in the doxorubicin -treated and thymoquinone -treated groups was more than double compared to the control group (p < 0.05; Fig. 2 ). Doxorubicin-thymoquinone combination led to potent apoptosis in cancer cells, in comparison with either doxorubicin or thymoquinone alone. The combination of doxorubicin and thymoquinone demonstrated significantly higher efficacy in inducing apoptosis in cancer cells, surpassing the effects of either doxorubicin or thymoquinone alone (p < 0.05). This suggests a synergistic effect between the two treatments, highlighting that thymoquinone exposure in Caco-2 cells sensitized cells to doxorubicin-induced apoptosis. Additionally, combination of doxorubicin with thymoquinone led to significant increase in the expression levels of Bax and caspase-3 and also significant decrease in the expression levels of Bcl-2 in Caco-2 cells (Fig. 2 ). This increase in Bax and caspase-3 expression and decrease in Bcl-2 expression can be attributed to the synergistic effect of doxorubicin and thymoquinone. Doxorubicin is known to induce apoptosis by activating Bax and caspase-3 and inhibiting Bcl-2, which are key players in the apoptotic pathway. Thymoquinone, on the other hand, enhances the effect of doxorubicin (Fig. 2 ). Together, these two treatments work in tandem to promote apoptosis and inhibit cell survival in Caco-2 cells. 3.3. Proliferative genes are thymoquinone targets in interfering with DOX sensitivity in colorectal cancer Our results showed that treatment of Caco-2 cells with thymoquinone and doxorubicin in alone resulted in the significant modulation in the expression levels of proliferative genes including P21, P53, Cyc D1, Cdk4, Cdk6 and PCNA (p < 0.05). P21 and P53 are two main tumor suppressor genes with major anti-proliferative functions in cells. We found that in cells exposed simultaneously to thymoquinone and doxorubicin, the mRNA and protein expression levels of P21 and P53 was upregulated in more extents in comparison to mono-treatments (P < 0.05). On the other hand, the mRNA and protein expression levels of Cyc D1, Cdk4, Cdk6 and PCNA was also found to be significantly lower in cells treated with combination of thymoquinone and doxorubicin. Therefore, these proteins are also suggested to be a target of thymoquinone (Fig. 3 ). Taking together, our results showed that thymoquinone increased doxorubicin sensitivity in colorectal cancer cells via downregulating Cyc D1, Cdk4, Cdk6 and PCNA and upregulating P21 and P53 (Fig. 3 ). To have in-depth investigation in thymoquinone mediated enhancement in doxorubicin sensitivity in Caco-2 cells, we knock down P21 via specific siRNA. Silencing of P21 via siRNA led to significant abolishment in the combination mediated suppression of proliferative genes including Cyc D1, Cdk4, Cdk6 and PCNA. Additionally, cells treated with si-P21 showed lower expression levels of P53. These results mean that P21 play key roles in the thymoquinone mediated doxorubicin chemosensitivity, such that P21 silencing completely abolished their effects on proliferative genes (Fig. 3 ). 3.4. Combination of thymoquinone and doxorubicin downregulated the PI3K/Akt axis in Caco-2 cells PI3K/Akt signaling is one of the main proliferative pathways that commonly showed aberrant expression profiles. We tested whether colorectal cancer cells treatment with combination of thymoquinone and doxorubicin exert any significant impact on the expression levels of key component of this signaling pathway including PI3K and Akt. For this purpose, qRT-PCR and western blotting were applied in cells treated thymoquinone and doxorubicin. The first important component of this signaling pathway is PI3K. In Caco-2 cells treated with combination of thymoquinone and doxorubicin, the mRNA and protein expression levels of PI3K were significantly lower than monotreatments (p < 0.05; Fig. 4 ). In addition, our results showed that in Caco-2 cells, thymoquinone and doxorubicin led to significant suppression of mRNA and protein expression levels of Akt, in comparison to monotreatments (p < 0.05; Fig. 4 ). Exposure of Caco-2 cells to si-P21 completely abolished the inhibitory effects of thymoquinone and doxorubicin combination on the PI3K/Akt signaling. 4. Discussion The results of this study indicate that natural thymoquinone significantly enhances the chemosensitivity of Caco-2 colorectal cancer cells to doxorubicin. Thymoquinone treatment resulted in a marked decrease in cell proliferation and an increase in cell death, as evidenced by the MTT assay and cell death ELISA. The molecular mechanisms underlying this enhanced chemosensitivity involve the upregulation of p21 and p53, as well as the downregulation of Cyclin D1, CDK4, CDK6, and PCNA. Additionally, thymoquinone suppressed the PI3K/AKT signaling pathway, which is known to play a critical role in cell survival and proliferation. The findings align with previous research suggesting that thymoquinone exhibits potent anticarcinogenic activity against various human malignancies. Several studies have demonstrated that thymoquinone possesses significant anticancer properties in colorectal cancer models ( 12 ). Research has shown that thymoquinone can induce apoptosis and inhibit tumor growth in animal models of colorectal cancer ( 13 – 15 ). Moreover, it has been observed to enhance the efficacy of standard chemotherapy drugs, making it a promising candidate for combination therapy in colorectal cancer treatment. In HCT-116 cells, Kundo et al. showed that thymoquinone decreased the expression of antiapoptotic proteins Bcl-2 and Bcl-xl, while amplifying Bax expression ( 16 ). Ballot et al. reported that thymoquinone exerted a potent anti-proliferative effect on colorectal cancer stem-like cells via inducing apoptosis and inhibiting NF-κB and MEK signaling ( 17 ). Muhtasib et al. showed that the anti-proliferative and pro-apoptotic effects of thymoquinone are mediated via induction of p53 ( 18 ). In addition, the observed effects on the PI3K/AKT pathway and cell cycle regulators highlight the potential of thymoquinone as a chemosensitizing agent ( 19 ). By targeting these key molecular pathways, thymoquinone enhances the cytotoxic effects of doxorubicin, thereby improving its efficacy in colorectal cancer treatment. The PI3K/Akt signaling pathway is frequently implicated in the progression of CRC ( 20 ). It has been observed that thymoquinone inhibits the activation of PI3K/Akt in colorectal cancer cell lines, such as HCT-116 and SW480, most likely through an increase in the PTEN tumor suppressor ( 9 , 20 , 21 ). These effects of thymoquinone are reported to result in the alteration of metabolic reprogramming in colorectal cancer cells, via suppressing key enzymes and factors involved in glycolysis and the Warburg effect ( 9 , 20 ). By thymoquinone-mediated suppression of the PI3K-AKT/HK2 pathway, CRC cells are less capable of tumorigenesis, including wound healing and invasion ( 9 ). The study also demonstrates the importance of p21 in mediating the effects of thymoquinone ( 22 ). P21 is a critical regulator of the cell cycle and apoptosis, and its upregulation in response to thymoquinone treatment suggests a pivotal role in enhancing doxorubicin-induced cytotoxicity ( 23 ). Furthermore, the suppression of the PI3K/AKT pathway by thymoquinone indicates a potential avenue for overcoming chemoresistance, as this pathway is frequently activated in cancer cells and contributes to their survival and proliferation. In conclusion, this study provides compelling evidence that thymoquinone can significantly enhance the chemosensitivity of colorectal cancer cells to doxorubicin by targeting p21 and the PI3K/AKT signaling pathway. These findings underscore the potential therapeutic benefits of thymoquinone in combination with conventional chemotherapeutic agents, offering a promising strategy for improving the outcomes of colorectal cancer treatment. Further in vivo studies and clinical trials are warranted to validate these findings and explore the full therapeutic potential of thymoquinone in cancer chemotherapy. Declarations Author Contribution P.Z: MethodologyS.L: Investigation P.Z:Writing - Original Draft Y.Z:Writing - Review & Editing & Supervision All authors reviewed the results and approved the final version of the manuscript. References Biller LH, Schrag D (2021) Diagnosis and treatment of metastatic colorectal cancer: a review. JAMA 325(7):669–685 Alzahrani SM, Al Doghaither HA, Al-Ghafari AB (2021) General insight into cancer: An overview of colorectal cancer. 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Molecules 29(24):5907 Karim S, Burzangi AS, Ahmad A, Siddiqui NA, Ibrahim IM, Sharma P et al (2022) PI3K-AKT pathway modulation by thymoquinone limits tumor growth and glycolytic metabolism in colorectal cancer. Int J Mol Sci 23(4):2305 Stefani C, Miricescu D, Stanescu-Spinu I-I, Nica RI, Greabu M, Totan AR et al (2021) Growth factors, PI3K/AKT/mTOR and MAPK signaling pathways in colorectal cancer pathogenesis: where are we now? Int J Mol Sci 22(19):10260 Kurowska N, Madej M, Strzalka-Mrozik B, Thymoquinone (2023) A Promising Therapeutic Agent for the Treatment of Colorectal Cancer. Curr Issues Mol Biol 46(1):121–139 Thabet NA, El-Khouly D, Sayed‐Ahmed MM, Omran MM (2021) Thymoquinone chemosensitizes human colorectal cancer cells to imatinib via uptake/efflux genes modulation. Clin Exp Pharmacol Physiol 48(6):911–920 Gali-Muhtasib H, Ocker M, Kuester D, Krueger S, El‐Hajj Z, Diestel A et al (2008) Thymoquinone reduces mouse colon tumor cell invasion and inhibits tumor growth in murine colon cancer models. J Cell Mol Med 12(1):330–342 Kensara OA, El-Shemi AG, Mohamed AM, Refaat B, Idris S, Ahmad J (2016) Thymoquinone subdues tumor growth and potentiates the chemopreventive effect of 5-fluorouracil on the early stages of colorectal carcinogenesis in rats. Drug design, development and therapy. :2239-53 Asfour W, Almadi S, Haffar L (2013) Thymoquinone suppresses cellular proliferation, inhibits VEGF production and obstructs tumor progression and invasion in the rat model of DMH-induced colon carcinogenesis Mottaghipisheh J, Taghrir H, Boveiri Dehsheikh A, Zomorodian K, Irajie C, Mahmoodi Sourestani M et al (2021) Linarin, a glycosylated flavonoid, with potential therapeutic attributes: A comprehensive review. Pharmaceuticals 14(11):1104 Ballout F, Monzer A, Fatfat M, El Ouweini H, Jaffa MA, Abdel-Samad R et al (2020) Thymoquinone induces apoptosis and DNA damage in 5-Fluorouracil-resistant colorectal cancer stem/progenitor cells. Oncotarget 11(31):2959 Gali-Muhtasib H, Diab-Assaf M, Boltze C, Al-Hmaira J, Hartig R, Roessner A et al (2004) Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53-dependent mechanism. Int J Oncol 25(4):857–866 Afrose SS, Junaid M, Akter Y, Tania M, Zheng M, Khan MA (2020) Targeting kinases with thymoquinone: A molecular approach to cancer therapeutics. Drug Discovery Today 25(12):2294–2306 Farrash WF, Aslam A, Almaimani R, Minshawi F, Almasmoum H, Alsaegh A et al (2023) Metformin and thymoquinone co-treatment enhance 5‐fluorouracil cytotoxicity by suppressing the PI3K/mTOR/HIF1α pathway and increasing oxidative stress in colon cancer cells. BioFactors 49(4):831–848 Hsu H-H, Chen M-C, Day CH, Lin Y-M, Li S-Y, Tu C-C et al (2017) Thymoquinone suppresses migration of LoVo human colon cancer cells by reducing prostaglandin E2 induced COX-2 activation. World J Gastroenterol 23(7):1171 Diab-Assaf M, Semaan J, El-Sabban M, Al Jaouni SK, Azar R, Kamal MA et al (2018) Inhibition of proliferation and induction of apoptosis by thymoquinone via modulation of TGF family, p53, p21 and Bcl-2α in leukemic cells. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents). 18(2):210–215 Ozturk SA, Alp E, Saglam ASY, Konac E, Menevse ES (2018) The effects of thymoquinone and genistein treatment on telomerase activity, apoptosis, angiogenesis, and survival in thyroid cancer cell lines. J Cancer Res Ther 14(2):328–334 Additional Declarations No competing interests reported. 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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-5941352","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":410551122,"identity":"e5d7723d-9463-472c-9e06-8dfcf157a86b","order_by":0,"name":"Peng Zhang","email":"","orcid":"","institution":"Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Zhang","suffix":""},{"id":410551123,"identity":"3d6817e4-448e-49cb-b52d-5a688a9ce27d","order_by":1,"name":"Shaowen Li","email":"","orcid":"","institution":"Affiliated Hospital of Hebei University","correspondingAuthor":false,"prefix":"","firstName":"Shaowen","middleName":"","lastName":"Li","suffix":""},{"id":410551124,"identity":"f054d0ad-1f4f-44e6-bcd0-c99b7131def9","order_by":2,"name":"Pei Zhang","email":"","orcid":"","institution":"Affiliated Hospital of Hebei University","correspondingAuthor":false,"prefix":"","firstName":"Pei","middleName":"","lastName":"Zhang","suffix":""},{"id":410551125,"identity":"02dc7d7c-e3f2-441e-aef8-9d9695012a8d","order_by":3,"name":"Yuanfang Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYBADfiBmfPChAkgxMzcQpUUSqIzZcMYZkBZG4rWwSfO2gdgEtMjPyD0mzVNzR4J/dvsFyZnzaqP524FaflRsw6nF4EZemjTPsWcSEnfOFBh83HY8d8ZhxgbGnjO3cWuRyDGT5mE7XMdwIychcea2Y7kNQC3MjG24tcjPAGn5d1hCHqjlMO+cY7nzCWkBGm4G9PVhCYMb6QebeRtqcjcQ0mJw5o2x5dy+wxKGN3KYGWccO5C7EajlID6/yLfnGN548+2whNyN9Oc/PtTU5c47f/jggx8VeBzGwMAiAaF5DIDEYTDzAD71QMD8AUKzPwASdQQUj4JRMApGwUgEABMxYOxwEQu/AAAAAElFTkSuQmCC","orcid":"","institution":"Affiliated Hospital of Hebei University","correspondingAuthor":true,"prefix":"","firstName":"Yuanfang","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2025-02-01 10:53:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5941352/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5941352/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75501038,"identity":"68fa59ce-d409-4b6a-bca3-5a5fe100e647","added_by":"auto","created_at":"2025-02-05 09:01:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25436,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of doxorubicin, thymoquinone and their combination on cell viability of Caco-2 cells\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5941352/v1/06bad055813b86b61bafe168.png"},{"id":75501042,"identity":"d39330c1-9740-41ce-bc9c-230fd4047d6b","added_by":"auto","created_at":"2025-02-05 09:01:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":73513,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of thymoquinone on doxorubicin mediated apoptosis. A) thymoquinone impact on doxorubicin mediated apoptosis rate of cells, B, C, D) the effects of thymoquinone and doxorubicin on expression levels of apoptotic markers.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5941352/v1/edca4e7c4e15748847f7df64.png"},{"id":75501039,"identity":"0cb7837a-9849-4392-92e9-ae484a93d34e","added_by":"auto","created_at":"2025-02-05 09:01:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":109302,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of thymoquinone and doxorubicin on expression levels of proliferative markers.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5941352/v1/77e40af562ee957fe53bce82.png"},{"id":75501040,"identity":"4eb8254c-a6ad-477a-8439-78e512b9b6c9","added_by":"auto","created_at":"2025-02-05 09:01:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":21759,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of thymoquinone and doxorubicin on expression levels of PI3K/Akt signaling\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5941352/v1/09da65bede5fad08955a71c4.png"},{"id":75607445,"identity":"c4b67f2f-ac12-4427-b479-76d473b1621a","added_by":"auto","created_at":"2025-02-06 09:39:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":510516,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5941352/v1/417bf016-c8b8-4c22-9aeb-e671d53c9e2c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Thymoquinone inhibits tumor progression and promotes chemo-sensitivity via modulation of P21/PI3K/Akt axis in colorectal cancer cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eColorectal cancer is one of the most prevalent cancers globally, with a significant impact on public health. It is the third most commonly diagnosed cancer in men and the second in women (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The incidence rates vary widely across regions, with the highest rates observed in developed countries, while lower rates are found in Africa and South-Central Asia (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). This variation is largely attributed to differences in dietary habits, lifestyle factors, and the availability of screening programs. Additionally, genetic predispositions, dietary factors, and lifestyle choices such as smoking, alcohol consumption, and physical inactivity contribute to the risk. Public health initiatives focusing on awareness, early detection, and lifestyle modifications are critical in reducing the global burden of colorectal cancer (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Early detection and treatment are crucial for improving survival rates, as advanced stages of colorectal cancer can spread to other parts of the body. Regular screening and awareness are essential for early diagnosis and effective management of this condition (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNatural compounds have gained attention in the treatment of colorectal cancer due to their potential therapeutic benefits and lower toxicity compared to conventional chemotherapy (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Phytochemicals such as flavonoids, polyphenols, and alkaloids from fruits, vegetables, and herbal medicines have shown potential in modulating signaling pathways involved in colorectal cancer progression (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). These natural compounds offer a promising avenue for developing novel therapeutic strategies, either as standalone treatments or in combination with existing therapies, to improve outcomes for colorectal cancer patients. Thymoquinone, a bioactive compound derived from black cumin seeds (Nigella sativa), has shown significant promise in the treatment of colorectal cancer (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). It exhibits potent anticancer activities, including the induction of apoptosis, inhibition of cell proliferation, and suppression of metastasis (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). It has shown significant promise in the treatment of colorectal cancer by modulating various signaling pathways, including the PI3K/AKT pathway (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The PI3K/AKT pathway is frequently activated in colorectal cancer and plays a crucial role in promoting cell survival, proliferation, and resistance to apoptosis (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Thymoquinone has been found to inhibit the PI3K/AKT pathway, leading to reduced tumor growth and enhanced chemosensitivity (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). By suppressing AKT phosphorylation, thymoquinone can induce apoptosis and inhibit the proliferation of colorectal cancer cells. This inhibition of the PI3K/AKT pathway also contributes to the suppression of metastasis, further highlighting the therapeutic potential of thymoquinone in colorectal cancer treatment (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe ability of thymoquinone to target the PI3K/AKT signaling pathway underscores its potential as a therapeutic agent, either as a standalone treatment or in combination with existing therapies, to improve outcomes for colorectal cancer patients. Further research is needed to fully elucidate the mechanisms by which thymoquinone modulates this pathway and its clinical applications. Therefore, this study is aimed to evaluate the effects of thymoquinone on the increasing doxorubicin chemosensitivity via targeting P21 and PI3K/AKT signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"2. Material and methods","content":"\u003cp\u003e2.1.Cell culture and transfection\u003c/p\u003e\u003cp\u003eWe obtained the Caco-2 cells from the Pastor Institute cell bank for the study of colorectal cancer (Tehran, Iran). RPMI-1640 cell culture medium containing 10% fetal bovine serum (FBS) and 100 U/mL antibiotic solution (penicillin/ streptomycin) was used to maintain and grow the cells. After reaching to exponential growth condition, cells were transfected with Lipofectamine™2000 reagent containing scrambled negative controls (si-NC) and p21 siRNA (si-p21) purchased from BioSciences co.\u003c/p\u003e\u003cp\u003e2.2. Evaluation of cell viability\u003c/p\u003e\u003cp\u003eIn order to determine the viability of Caco-2 cells, 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) was used as a staining method. 96-well plates were used to culture the cells and then treated with doxorubicin or thymoquinone at different concentrations. Incubation was extended for another 4 hours with fresh medium containing 20 µL MTT solution after 48 hours. To dissolve the formed crystals, 150 µL of DMSO were used. Three replicate experiments were conducted at 490 nm to measure absorbance.\u003c/p\u003e\u003cp\u003e2.3. qRT-PCR\u003c/p\u003e\u003cp\u003eFor measuring the mRNA expression levels of P21, P53, Cyc D1, Cdk4, Cdk6, PCNA, PI3K, Akt, Bax, Bcl-2, and capsase-3, qRT-PCR was performed. In all groups, total RNA was extracted from cells using a specific RNA isolation kit. RNA samples were analyzed for quality and quantity, and reverse transcription to cDNA was performed according to the manufacturer's instructions. q-PCR was applied by using the SYBR Green and specific primers on Rotor-Gene™ 6000 system (Corbett Life Science, Mortlake, Australia).\u003c/p\u003e\u003cp\u003e2.4. Western blotting\u003c/p\u003e\u003cp\u003ePreparation of suitable protein samples for western blotting was performed using RIPA buffer (Sigma-Aldrich). Protein concentration was also determined by the Bradford method, and proteins were separated by 10% SDS-polyacrylamide gel electrophoresis. After this step, PVDF membranes were used to transfer separated proteins. At room temperature, the membrane was then placed in a buffer containing 5% bovine serum albumin for 2 hours, and then polyclonal antibodies for P21, P53, Cyc D1, Cdk4, Cdk6, PCNA, PI3K, Akt, Bax, Bcl-2, capsase-3 and β-actin were added and incubated at 4ºc overnight. Subsequently, secondary antibodies were added and incubated for 2 h at room temperature. The final step was to expose and photograph the slide.\u003c/p\u003e\u003cp\u003e2.5. ELISA cell death assay\u003c/p\u003e\u003cp\u003eELISA cells death assay was applied to measure the apoptosis rate of Caco-2 cells treated with DOX and thymoquinone. All steps were followed in accordance to the manufacturer’s protocol.\u003c/p\u003e\u003cp\u003e2.6. Statistical analysis\u003c/p\u003e\u003cp\u003eAll data were expressed as mean ± SD. Kolmogorov-Smirnov test was used to assess normality of the data and one-way analysis of variance (ANOVA), followed Tukey’s test were performed for evaluation of multiple comparison using GraphPad prism software. The values were considered statistically significant at P \u0026lt; 0.05.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1. The effects of thymoquinone on the doxorubicin mediated inhibition of cells viability and proliferation in Caco-2 cells\u003c/p\u003e\u003cp\u003eMTT assay was performed to measure the cytotoxic effects of doxorubicin, thymoquinone, and their combination following Caco-2 cells exposure to these treatments. Upon 48-h incubation, cell viability was measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). By increasing the concentration of doxorubicin from 0 to 5 µM and thymoquinone from 0 to 800 µM, its inhibitory effect on cell viability was increased, such that the cells viability was almost completely suppressed in higher concentrations of doxorubicin and also thymoquinone. In Caco-2 cell line, doxorubicin IC50 was 2.4 ± 1 µM and thymoquinone IC50 was 470 ± 12 µM after treating the cells for 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, simultaneous exposure of Caco-2 cells to different amounts of doxorubicin and 470 µM of thymoquinone led to potentiation of doxorubicin mediated inhibition of cell viability, such that the IC50 value of doxorubicin was decreased from 2.4 to 1.2 µM.\u003c/p\u003e\u003cp\u003e3.2. Thymoquinone modulates Caco-2 cells' apoptotic response to doxorubicin\u003c/p\u003e\u003cp\u003eOur next step was to determine whether pairing doxorubicin and thymoquinone increased Caco-2 cell apoptosis. When cells were treated with doxorubicin or thymoquinone, the apoptosis rate was significantly higher than when they were not treated. This increase in apoptosis rate can be seen in the data, where the percentage of apoptotic cells in the doxorubicin -treated and thymoquinone -treated groups was more than double compared to the control group (p \u0026lt; 0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Doxorubicin-thymoquinone combination led to potent apoptosis in cancer cells, in comparison with either doxorubicin or thymoquinone alone. The combination of doxorubicin and thymoquinone demonstrated significantly higher efficacy in inducing apoptosis in cancer cells, surpassing the effects of either doxorubicin or thymoquinone alone (p \u0026lt; 0.05). This suggests a synergistic effect between the two treatments, highlighting that thymoquinone exposure in Caco-2 cells sensitized cells to doxorubicin-induced apoptosis. Additionally, combination of doxorubicin with thymoquinone led to significant increase in the expression levels of Bax and caspase-3 and also significant decrease in the expression levels of Bcl-2 in Caco-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This increase in Bax and caspase-3 expression and decrease in Bcl-2 expression can be attributed to the synergistic effect of doxorubicin and thymoquinone. Doxorubicin is known to induce apoptosis by activating Bax and caspase-3 and inhibiting Bcl-2, which are key players in the apoptotic pathway. Thymoquinone, on the other hand, enhances the effect of doxorubicin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Together, these two treatments work in tandem to promote apoptosis and inhibit cell survival in Caco-2 cells.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003e3.3. Proliferative genes are thymoquinone targets in interfering with DOX sensitivity in colorectal cancer\u003c/p\u003e\u003cp\u003eOur results showed that treatment of Caco-2 cells with thymoquinone and doxorubicin in alone resulted in the significant modulation in the expression levels of proliferative genes including P21, P53, Cyc D1, Cdk4, Cdk6 and PCNA (p \u0026lt; 0.05). P21 and P53 are two main tumor suppressor genes with major anti-proliferative functions in cells. We found that in cells exposed simultaneously to thymoquinone and doxorubicin, the mRNA and protein expression levels of P21 and P53 was upregulated in more extents in comparison to mono-treatments (P \u0026lt; 0.05). On the other hand, the mRNA and protein expression levels of Cyc D1, Cdk4, Cdk6 and PCNA was also found to be significantly lower in cells treated with combination of thymoquinone and doxorubicin. Therefore, these proteins are also suggested to be a target of thymoquinone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Taking together, our results showed that thymoquinone increased doxorubicin sensitivity in colorectal cancer cells via downregulating Cyc D1, Cdk4, Cdk6 and PCNA and upregulating P21 and P53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo have in-depth investigation in thymoquinone mediated enhancement in doxorubicin sensitivity in Caco-2 cells, we knock down P21 via specific siRNA. Silencing of P21 via siRNA led to significant abolishment in the combination mediated suppression of proliferative genes including Cyc D1, Cdk4, Cdk6 and PCNA. Additionally, cells treated with si-P21 showed lower expression levels of P53. These results mean that P21 play key roles in the thymoquinone mediated doxorubicin chemosensitivity, such that P21 silencing completely abolished their effects on proliferative genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e3.4. Combination of thymoquinone and doxorubicin downregulated the PI3K/Akt axis in Caco-2 cells\u003c/p\u003e\u003cp\u003ePI3K/Akt signaling is one of the main proliferative pathways that commonly showed aberrant expression profiles. We tested whether colorectal cancer cells treatment with combination of thymoquinone and doxorubicin exert any significant impact on the expression levels of key component of this signaling pathway including PI3K and Akt. For this purpose, qRT-PCR and western blotting were applied in cells treated thymoquinone and doxorubicin. The first important component of this signaling pathway is PI3K. In Caco-2 cells treated with combination of thymoquinone and doxorubicin, the mRNA and protein expression levels of PI3K were significantly lower than monotreatments (p \u0026lt; 0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In addition, our results showed that in Caco-2 cells, thymoquinone and doxorubicin led to significant suppression of mRNA and protein expression levels of Akt, in comparison to monotreatments (p \u0026lt; 0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Exposure of Caco-2 cells to si-P21 completely abolished the inhibitory effects of thymoquinone and doxorubicin combination on the PI3K/Akt signaling.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe results of this study indicate that natural thymoquinone significantly enhances the chemosensitivity of Caco-2 colorectal cancer cells to doxorubicin. Thymoquinone treatment resulted in a marked decrease in cell proliferation and an increase in cell death, as evidenced by the MTT assay and cell death ELISA. The molecular mechanisms underlying this enhanced chemosensitivity involve the upregulation of p21 and p53, as well as the downregulation of Cyclin D1, CDK4, CDK6, and PCNA. Additionally, thymoquinone suppressed the PI3K/AKT signaling pathway, which is known to play a critical role in cell survival and proliferation.\u003c/p\u003e\u003cp\u003eThe findings align with previous research suggesting that thymoquinone exhibits potent anticarcinogenic activity against various human malignancies. Several studies have demonstrated that thymoquinone possesses significant anticancer properties in colorectal cancer models (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Research has shown that thymoquinone can induce apoptosis and inhibit tumor growth in animal models of colorectal cancer (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e–\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Moreover, it has been observed to enhance the efficacy of standard chemotherapy drugs, making it a promising candidate for combination therapy in colorectal cancer treatment. In HCT-116 cells, Kundo et al. showed that thymoquinone decreased the expression of antiapoptotic proteins Bcl-2 and Bcl-xl, while amplifying Bax expression (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Ballot et al. reported that thymoquinone exerted a potent anti-proliferative effect on colorectal cancer stem-like cells via inducing apoptosis and inhibiting NF-κB and MEK signaling (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Muhtasib et al. showed that the anti-proliferative and pro-apoptotic effects of thymoquinone are mediated via induction of p53 (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition, the observed effects on the PI3K/AKT pathway and cell cycle regulators highlight the potential of thymoquinone as a chemosensitizing agent (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). By targeting these key molecular pathways, thymoquinone enhances the cytotoxic effects of doxorubicin, thereby improving its efficacy in colorectal cancer treatment. The PI3K/Akt signaling pathway is frequently implicated in the progression of CRC (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). It has been observed that thymoquinone inhibits the activation of PI3K/Akt in colorectal cancer cell lines, such as HCT-116 and SW480, most likely through an increase in the PTEN tumor suppressor (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). These effects of thymoquinone are reported to result in the alteration of metabolic reprogramming in colorectal cancer cells, via suppressing key enzymes and factors involved in glycolysis and the Warburg effect (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). By thymoquinone-mediated suppression of the PI3K-AKT/HK2 pathway, CRC cells are less capable of tumorigenesis, including wound healing and invasion (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe study also demonstrates the importance of p21 in mediating the effects of thymoquinone (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). P21 is a critical regulator of the cell cycle and apoptosis, and its upregulation in response to thymoquinone treatment suggests a pivotal role in enhancing doxorubicin-induced cytotoxicity (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Furthermore, the suppression of the PI3K/AKT pathway by thymoquinone indicates a potential avenue for overcoming chemoresistance, as this pathway is frequently activated in cancer cells and contributes to their survival and proliferation.\u003c/p\u003e\u003cp\u003eIn conclusion, this study provides compelling evidence that thymoquinone can significantly enhance the chemosensitivity of colorectal cancer cells to doxorubicin by targeting p21 and the PI3K/AKT signaling pathway. These findings underscore the potential therapeutic benefits of thymoquinone in combination with conventional chemotherapeutic agents, offering a promising strategy for improving the outcomes of colorectal cancer treatment. Further in vivo studies and clinical trials are warranted to validate these findings and explore the full therapeutic potential of thymoquinone in cancer chemotherapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eP.Z: MethodologyS.L: Investigation P.Z:Writing - Original Draft Y.Z:Writing - Review \u0026amp; Editing \u0026amp; Supervision All authors reviewed the results and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBiller LH, Schrag D (2021) Diagnosis and treatment of metastatic colorectal cancer: a review. JAMA 325(7):669\u0026ndash;685\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlzahrani SM, Al Doghaither HA, Al-Ghafari AB (2021) General insight into cancer: An overview of colorectal cancer. 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Int J Mol Sci 23(4):2305\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStefani C, Miricescu D, Stanescu-Spinu I-I, Nica RI, Greabu M, Totan AR et al (2021) Growth factors, PI3K/AKT/mTOR and MAPK signaling pathways in colorectal cancer pathogenesis: where are we now? Int J Mol Sci 22(19):10260\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurowska N, Madej M, Strzalka-Mrozik B, Thymoquinone (2023) A Promising Therapeutic Agent for the Treatment of Colorectal Cancer. Curr Issues Mol Biol 46(1):121\u0026ndash;139\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThabet NA, El-Khouly D, Sayed‐Ahmed MM, Omran MM (2021) Thymoquinone chemosensitizes human colorectal cancer cells to imatinib via uptake/efflux genes modulation. 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BioFactors 49(4):831\u0026ndash;848\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHsu H-H, Chen M-C, Day CH, Lin Y-M, Li S-Y, Tu C-C et al (2017) Thymoquinone suppresses migration of LoVo human colon cancer cells by reducing prostaglandin E2 induced COX-2 activation. World J Gastroenterol 23(7):1171\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiab-Assaf M, Semaan J, El-Sabban M, Al Jaouni SK, Azar R, Kamal MA et al (2018) Inhibition of proliferation and induction of apoptosis by thymoquinone via modulation of TGF family, p53, p21 and Bcl-2α in leukemic cells. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents). 18(2):210\u0026ndash;215\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOzturk SA, Alp E, Saglam ASY, Konac E, Menevse ES (2018) The effects of thymoquinone and genistein treatment on telomerase activity, apoptosis, angiogenesis, and survival in thyroid cancer cell lines. J Cancer Res Ther 14(2):328\u0026ndash;334\u003c/span\u003e\u003c/li\u003e\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":"[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":"Cisplatin, colorectal cancer, thymoquinone, siRNA, apoptosis, p21, P53","lastPublishedDoi":"10.21203/rs.3.rs-5941352/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5941352/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNatural thymoquinone has been extensively used as a chemopreventive agent and has shown potent anticarcinogenic activity against a broad range of human malignancies. However, the underlying mechanisms and involving signaling pathways are still not well studies. Therefore, this study is aimed to evaluate the effects of thymoquinone on the increasing doxorubicin chemosensitivity via targeting P21 and PI3K/AKT signaling. Caco-2 cells were treated with thymoquinone. MTT assay were applied to assess the impact of different dose of thymoquinone on the doxorubicin cytotoxicity. The mRNA and protein expression levels of PI3K, Akt, P21, P53, Cyc D1, Cdk4, Cdk6, PCNA, Bax, Bcl-2 and caspase-3 were assessed by qRT-PCR and western blotting. A cell death ELISA commercial kits were used to measure apoptosis. We found that thymoquinone treatment significantly decreased proliferation rate in Caco-2 colorectal cancer cells. The survival rate of cells was reduced significantly when doxorubicin was combined with thymoquinone. Thymoquinone upregulated p21, P53 and downregulated Cyc D1, Cdk4, Cdk6, PCNA, as well as suppressed PI3K/Akt signaling pathway. We conclude that thymoquinone induces doxorubicin sensitivity in colorectal cancer cells through targeting p21 and the PI3K/AKT pathway, thus implicating its importance in chemotherapy for colorectal cancer.\u003c/p\u003e","manuscriptTitle":"Thymoquinone inhibits tumor progression and promotes chemo-sensitivity via modulation of P21/PI3K/Akt axis in colorectal cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-05 09:01:05","doi":"10.21203/rs.3.rs-5941352/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":"c65ad3de-1921-47a3-8753-961e6d061ab8","owner":[],"postedDate":"February 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-06T09:38:46+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-05 09:01:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5941352","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5941352","identity":"rs-5941352","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}