Exploring the Efficacy of a Novel Cannabidiol and Bevacizumab Combination in Non-Small Cell Lung Cancer

Exploring the Efficacy of a Novel Cannabidiol and Bevacizumab Combination in Non-Small Cell Lung Cancer | 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 Exploring the Efficacy of a Novel Cannabidiol and Bevacizumab Combination in Non-Small Cell Lung Cancer Abdul Qadir, Zurqa Khalid, Ayesha Kashmala Ghauri, Muhammad Hamza Dawood, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7737896/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Mar, 2026 Read the published version in DARU Journal of Pharmaceutical Sciences → Version 1 posted You are reading this latest preprint version Abstract Background Lung cancer is the leading cause of cancer-related deaths worldwide. Despite advancements in cancer treatment, the survival rate for patients with non-small cell lung cancer (NSCLC) remains around 15%. Therefore, new therapeutic options are needed for treating the patients with NSCLC. Objectives The study's objective was to investigate the effect of the cannabidiol and bevacizumab combination against the human NSCLC and its underlying molecular mechanism using A549 cancer cell line as an in vitro model. Methods The A549 cells were cultured and treated with cannabidiol, bevacizumab, and their combination. After incubation, cell growth was evaluated using the MTT assay, and cell apoptosis was determined using flowcytometry. The impact on apoptotic genes was assessed through western blotting, whereas the expression levels of tumor metastasis genes were analyzed using qPCR. Results The proliferation of A549 cells was inhibited in a dose-dependent manner by cannabidiol, bevacizumab, and their combination. The apoptotic rate was 14.1% with cannabidiol and 26.5% with bevacizumab; however, when the two drugs were used in combination, the rate of apoptosis was significantly increased by 52.2%, respectively (p < 0.001). The underlying mechanism of apoptosis was further elaborated by western blotting, which displayed that co-treatment downregulated considerably the anti-apoptotic gene Bcl-2, and upregulated the pro-apoptotic gene caspase-9 (p < 0.001). Moreover, in comparison to the individual drug, the co-treatment dramatically decreased the expression of MMP-2 and MMP-9 (p < 0.001). Conclusion The combination of cannabidiol and bevacizumab has a very potent anticancer effect on the growth inhibition of NSCLC than using either drug alone. Apoptosis Bevacizumab Cannabidiol MMP-2 MMP-9 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Lung cancer is among the most serious health problems and is thought to be the most frequent cause of mortality linked to cancer, despite advances in medical science [1]. Lung cancer causes more deaths as compared to breast, colon, or prostate cancer. It is of two types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC constitutes approximately 85% of all kinds of lung cancer; on the other hand, SCLC makes up about 15% in total [2]. Despite breakthroughs in the management of cancer, the 5-year survival rate among patients with NSCLC is 15%. Therefore, in order to treat patients with NSCLC, new therapeutic alternatives are required [1] . Matrix metalloproteinases (MMPs) are members of the proteolytic enzyme family. that are primarily engaged in the breakdown of the extracellular matrix and stimulate invasion of tumor and metastasis [3]. The MMP family comprises 23 members, and among these, MMP-2 and − 9 are regarded as pivotal in the development of tumors since they are primarily expressed in multiple human malignancies: stomach, prostate, bladder, ovarian, lung and breast cancer. They are regarded as key mediators of tumor growth and metastasis; therefore, it has been assumed that inhibiting MMP-2 and MMP-9 is an ideal treatment for cancer [4–6] . Bevacizumab is a monoclonal antibody approved by the FDA for treating various human cancers. It acts predominantly by targeting VEGF in order to stop tumor growth and metastasis [7–9]. It is widely used as first-line therapy for tumors in their intermediate and advanced stages, including renal cell carcinoma, glioblastoma, NSCLC, and colorectal cancer [8,10,11]. Additionally, off-label use is employed to treat iris neovascularization, age-related macular degeneration, and diabetic retinopathy [12] . Cannabidiol belongs to the family of cannabinoids and is derived from cannabis sativa. It is an inverse agonist on the CB2 receptor and an antagonist on the CB1 receptor. Moreover, it works as an agonist at PPAR, 5HT1A, and TRP receptors and has also been observed to impede vascular endothelial cell survival and migration [13–15]. Although it is primarily used to treat anxiety and depression [16], multiple studies have revealed that it is also effective against a number of cancers, such as prostate cancer, breast and gastric cancers also gliomas [13,14,17] . The aim of the present study is to explore the impact of the cannabidiol and bevacizumab combination on NSCLC growth inhibition and its underlying molecular mechanism. 2. Methods 2.1. Cell Culture The A549 cell line was acquired from the American Type Culture Collection. In RPMI media, the cells were cultured supplemented with 10% FBS and penicillin/streptomycin (100 U/mL). A cell culture incubator with 5% CO 2 and 95% humidified air was used to maintain the cells at 37°C. 2.2. MTT assay The MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide} was used to observe the drugs effect on the proliferation of A549 cancer cells. Approximately 10,000 cells were added to a 96-well plate. Following a 24-hour incubation period, they were treated with cannabidiol (Epidiolex), bevacizumab (Avastin), and their combination. At the end of the 48 hours of treatment, 0.5% MTT solution was added, and the absorbance was recorded using a spectrophotometer at a 560 nm wavelength. 2.3. Flowcytometry analysis Flowcytometry (BD FACS Celesta) was used for analyzing cell apoptosis. Cells that had reached 80% confluence were treated with cannabidiol, bevacizumab, and their combination for 48 hours. Subsequently, after centrifugation cells were collected and resuspended in 500µl of binding buffer and allowed to incubate for a duration of 15 minutes at room temperature. Followed by addition of 5 µl each of annexin V-FITC and propidium iodide, the data was subjected to flow cytometry analysis with FACSDiva Software 8.0. 2.4 Western blot analysis The effect on the apoptotic genes was investigated using western blotting. Cannabidiol, bevacizumab, and their combination were administered to the A549 cancer cells for a 48-hour period. Following the collection of the cells using RIPA buffer, the total protein concentration was measured via the Pierce Biotechnology BCA Protein Assay Kit. For electrophoresis, 50 µg of proteins were run through a 10% SDS-PAGE. The western blot was performed using primary antibodies, caspase-9, Bcl-2, GAPDH and secondary antibodies, IgG-horseradish peroxidase. Elecsys-2010 (Roche Professional Diagnostics) was used to collect the data, and ImageJ software 1.52 was used to calculate the optical densities for each band. 2.5 Quantitative real-time polymerase chain reaction (qPCR) RNA extraction was executed by TRIzol reagent after treating the cells with cannabidiol, bevacizumab, and their combination. About 1 µg of RNA was then reverse transcribed, and the MMP-2 & -9 mRNA levels were determined with the Qiagen SYBR Green PCR Master Mix using qPCR. GAPDH was used as a reference gene for normalization. The primer sequences used for this method are: MMP-2: 5’-CAGGCTCTTCTCCTTTCACAAC-3’ and 5’-AAGCCACGGCTTGGTTTTCCTC-3’; MMP-9: 5’-TGGGCTACGTGACCTATGACAT-3’ and 5’-GCCCAGCCCACCTCCACTCCTC-3’; GAPDH: 5’-TGACTTCAACAGCGACACCCA-3’ and 5’-CACCCTGTTGCTGTAGCCAAA-3’. 2.6 Statistical Analysis SPSS version 22 software for statistical analysis was utilized to analyze the data. ANOVA and Tukey’s post hoc test were used in comparison of the control and treatment groups. P-values below 0.05 were regarded as statistically significant. 3. Results 3.1. Effect on proliferation of A549 lung cancer cells A549 lung cancer cell growth was inhibited by cannabidiol by 8.6%, 15.4%, and 29.2% at 7.5 µM, 15 µM, and 30 µM. A549 lung cancer cell growth was suppressed by bevacizumab by 11.1%, 18.5%, and 33.3% at 4 µM, 8 µM, and 16 µM, respectively. The combination of cannabidiol and bevacizumab inhibited A549 lung cancer cell growth by 22%, 36.8%, and 67.3%, respectively. These findings demonstrated a concentration-dependent decline in A549 lung cancer cell proliferation with a noticeable reduction in cell proliferation when the two drugs were combined together, as seen in Fig. 1 . 3.2. Effect on A549 lung cancer cells apoptosis Flowcytometry examination revealed single-drug treatment, and the rate of apoptosis was 15.2% with cannabidiol and 21.9% with bevacizumab, respectively. When both medications were used together, the rate of apoptosis rose to 50.4% in comparison to the control group, as shown in Fig. 2 . These results suggest that treating lung cancer with a drug combination has significant therapeutic potential. 3.3. Effect on apoptotic genes in A549 lung cancer cells The Western blot findings exhibited a comparison of apoptotic genes in cannabidiol and bevacizumab groups; the anti-apoptotic gene (Bcl-2) expression in the group that received both cannabidiol and bevacizumab was significantly lower (p < 0.001). Additionally, compared to the cannabidiol and bevacizumab groups, the expression of pro-apoptotic genes (caspase-9) was considerably higher in the cannabidiol + bevacizumab group, as shown in Fig. 3 . These findings imply that co-treating A549 lung cancer cells with cannabidiol and bevacizumab increased the cells apoptotic response via both pathways. 3.4. Effect on MMP genes in A549 lung cancer cells Results of the qPCR demonstrated that cannabidiol and bevacizumab both downregulated MMP-2 and − 9 expression in A549 lung cancer cells; however, the combination of the two drugs exhibited a more pronounced reduction than either drug alone (p < 0.001), as shown in Fig. 4 . 4. Discussion The treatment of NSCLC has changed significantly during the past ten years. The FDA has approved bevacizumab to treat patients with mid- and advanced-stage NSCLC [8]. Cannabidiol, on the other hand, is not typically used to treat NSCLC patients, but studies have shown that it has anticancer properties in addition to being a cannabinoid and has the ability to cause cancer cells to undergo apoptosis [18]. Therefore, utilizing A549 cells of lung cancer as an in vitro model, the aim of the current research was to examine if the addition of cannabidiol would enhance the anticancer effect of bevacizumab against NSCLC. Using the MTT assay, we first looked at how cannabidiol, bevacizumab, and their combination affected the growth of lung cancer cells. Figure 5 Our findings demonstrated that the A549 cell growth was inhibited in a concentration-dependent manner, and these results aligned with the research that Kim et al and Wang et al had reported [2,8]. Our findings also revealed that when two drugs were taken together, the inhibition was more noticeable than when they were taken separately. Apoptosis, commonly referred to as programmed cell death or cell suicide, is a genetically induced process of cell self-destruction. It is important for the growth and control of tissue homeostasis. It occurs when there is an imbalance between anti-apoptotic and pro-apoptotic proteins [19]. Therefore, we evaluated changes in pro-apoptotic proteins (caspase-9) & anti-apoptotic proteins (Bcl-2) in cannabidiol, bevacizumab, and cannabidiol + bevacizumab-treated A549 cells. Our results revealed that, in contrast to the single-drug approach, the group receiving cannabidiol + bevacizumab had significantly lower Bcl-2 expression and higher caspase-9 expression than the other groups (p < 0.001) [2,8,9,20]. These findings confirm that the cannabidiol and bevacizumab combination induces apoptosis in A549 lung cancer cells. Last but not least, we examined the effects of cannabidiol and bevacizumab on MMP-2 and − 9, which are crucial for tumor growth and invasion and are expressed in various human cancers [16,21]. According to our findings, cannabidiol and bevacizumab both individually decreased the MMP-2 and − 9 expression levels. These results are consistent with other research that has been published [9,16]. Compared to the single-drug treatment, there was a greater suppression of MMP-2 and MMP-9 expression when cannabidiol and bevacizumab were used in combination. Due to a lack of funds, we used only one non-small cell lung cancer cell line (A549 cells) for evaluating the effect of the drugs and their underlying molecular mechanism. 5. Conclusion This study shows that co-treatment of cannabidiol and bevacizumab has a more prominent effect on the growth inhibition of NSCLC than bevacizumab alone. However, to establish the combined therapeutic benefits of these two drugs, a thorough clinical trial is necessary. Declarations Ethical approval: This study was performed in line with the principles of the Declaration of Helsinki. Ethical approval was obtained from the Institutional review board of Dow University of Health Sceinces, Karachi, Pakistan. (IRB-3274/DUHS/EXEMPTION/2023/460) Consent to participate: Not applicable since study utilized cell line. Consent to publish: Not applicable. Data Sharing Statement Data is not publicly available due to confidentiality but are available upon request to the corresponding author in de-identifiable form. Funding: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contribution Conceptualization: Abdul Qadir, Zurqa Khalid; Methodology: Abdul Qadir, Ayesha Kashmala Ghauri; Formal analysis and investigation: Abdul Qadir, Muhammad Hamza Dawood ; Writing - original draft preparation: Abdul Qadir, Zurqa Khalid, Ayesha Kashmala Ghauri, Muhammad Hamza Dawood, Usama Manzoor, Habib Ur Rehman; Writing - review and editing: Abdul Qadir, Zurqa Khalid, Ayesha Kashmala Ghauri, Muhammad Hamza Dawood, Usama Manzoor, Habib Ur Rehman; Resources: Abdul Qadir, Zurqa Khalid; Supervision: Abdul Qadir, Zurqa Khalid. Acknowledgement None. 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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-7737896","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":540377619,"identity":"4623b822-d17e-4231-aff4-a594cd42b76d","order_by":0,"name":"Abdul Qadir","email":"","orcid":"","institution":"United Medical and dental College","correspondingAuthor":false,"prefix":"","firstName":"Abdul","middleName":"","lastName":"Qadir","suffix":""},{"id":540377621,"identity":"85c3c29c-cdb0-4761-8108-58646b9dfd6f","order_by":1,"name":"Zurqa Khalid","email":"","orcid":"","institution":"Dow University of Health 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1","display":"","copyAsset":false,"role":"figure","size":160511,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of (A) cannabidiol (B) bevacizumab (C) and their combination on growth inhibition of A549 lung cancer cells. Data are expressed as the mean ± standard deviation of three experiments.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7737896/v1/cd34905ae87abf52e1ee37e0.jpeg"},{"id":95565106,"identity":"b8c01356-2c59-4d67-bd63-57ed427dc00e","added_by":"auto","created_at":"2025-11-10 16:12:35","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":147571,"visible":true,"origin":"","legend":"\u003cp\u003eThe Flowcytometric analysis showing (A) the effect of cannabidiol, bevacizumab and their combination on cell apoptosis in A549 lung cancer cells. (B) Showing the rate of apoptosis in A549 lung cancer cells. Data are expressed as the mean ± standard deviation of three experiments.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7737896/v1/a535dc0edb54c463688b1382.jpeg"},{"id":95655740,"identity":"5c02569d-907c-4687-8d41-bba8fb186d42","added_by":"auto","created_at":"2025-11-11 16:16:50","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":453886,"visible":true,"origin":"","legend":"\u003cp\u003eThe western blot analysis showing (A) the effect of cannabidiol (CBD), bevacizumab (BEV) and their combination (CBD + BEV) on expression of apoptotic genes (Bcl-2 and Caspase-9) in A549 lung cancer cells. (B-C) showing the quantification of Bcl-2 and caspase-9. Each bar represents the mean ± SD of three independent experiments. *p\u0026lt;0.05 as compared to control (n=3).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7737896/v1/13f96dfecee34a3cab979ca2.jpeg"},{"id":95655682,"identity":"db9b1323-6089-4233-997d-02ae059151c8","added_by":"auto","created_at":"2025-11-11 16:16:42","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":90442,"visible":true,"origin":"","legend":"\u003cp\u003eThe qPCR analysis (A-B) showing the effect of cannabidiol, bevacizumab and their combination on MMP-2 and MMP-9 expression in A549 lung cancer cells. Data are expressed as the mean ± standard deviation of three independent experiments. *p\u0026lt;0.05 as compared to control (n=3).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7737896/v1/3fa15159a5189318143c7660.jpeg"},{"id":95565105,"identity":"e519d34c-60b8-429a-8d57-a170c654f167","added_by":"auto","created_at":"2025-11-10 16:12:35","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":139680,"visible":true,"origin":"","legend":"\u003cp\u003eCentral Illustration of Efficacy of Cannabidiol and Bevacizumab Combination in Non-Small Cell Lung Cancer.\u003c/p\u003e\n\u003cp\u003eCannabidiol and bevacizumab inhibited A549 cell proliferation dose-dependently. Their combination significantly increased apoptosis (52.2%, p\u0026lt;0.001), suppressed Bcl-2, elevated caspase-9, and reduced MMP-2/9 expression compared to single treatments, highlighting synergistic pro-apoptotic and anti-invasive effects.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7737896/v1/a4614ed61eaf9b33de52ca55.jpeg"},{"id":104251081,"identity":"ca8a3d1c-53e2-452c-bdc4-206873b48833","added_by":"auto","created_at":"2026-03-09 16:11:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1576027,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7737896/v1/6dd86c6c-03ed-44e7-9243-0ce901d8e0d9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring the Efficacy of a Novel Cannabidiol and Bevacizumab Combination in Non-Small Cell Lung Cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLung cancer is among the most serious health problems and is thought to be the most frequent cause of mortality linked to cancer, despite advances in medical science [1]. Lung cancer causes more deaths as compared to breast, colon, or prostate cancer. It is of two types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC constitutes approximately 85% of all kinds of lung cancer; on the other hand, SCLC makes up about 15% in total [2]. Despite breakthroughs in the management of cancer, the 5-year survival rate among patients with NSCLC is 15%. Therefore, in order to treat patients with NSCLC, new therapeutic alternatives are required [1] .\u003c/p\u003e\u003cp\u003eMatrix metalloproteinases (MMPs) are members of the proteolytic enzyme family. that are primarily engaged in the breakdown of the extracellular matrix and stimulate invasion of tumor and metastasis [3]. The MMP family comprises 23 members, and among these, MMP-2 and \u0026minus;\u0026thinsp;9 are regarded as pivotal in the development of tumors since they are primarily expressed in multiple human malignancies: stomach, prostate, bladder, ovarian, lung and breast cancer. They are regarded as key mediators of tumor growth and metastasis; therefore, it has been assumed that inhibiting MMP-2 and MMP-9 is an ideal treatment for cancer [4\u0026ndash;6] .\u003c/p\u003e\u003cp\u003eBevacizumab is a monoclonal antibody approved by the FDA for treating various human cancers. It acts predominantly by targeting VEGF in order to stop tumor growth and metastasis [7\u0026ndash;9]. It is widely used as first-line therapy for tumors in their intermediate and advanced stages, including renal cell carcinoma, glioblastoma, NSCLC, and colorectal cancer [8,10,11]. Additionally, off-label use is employed to treat iris neovascularization, age-related macular degeneration, and diabetic retinopathy [12] .\u003c/p\u003e\u003cp\u003eCannabidiol belongs to the family of cannabinoids and is derived from cannabis sativa. It is an inverse agonist on the CB2 receptor and an antagonist on the CB1 receptor. Moreover, it works as an agonist at PPAR, 5HT1A, and TRP receptors and has also been observed to impede vascular endothelial cell survival and migration [13\u0026ndash;15]. Although it is primarily used to treat anxiety and depression [16], multiple studies have revealed that it is also effective against a number of cancers, such as prostate cancer, breast and gastric cancers also gliomas [13,14,17] .\u003c/p\u003e\u003cp\u003eThe aim of the present study is to explore the impact of the cannabidiol and bevacizumab combination on NSCLC growth inhibition and its underlying molecular mechanism.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Cell Culture\u003c/h2\u003e\u003cp\u003eThe A549 cell line was acquired from the American Type Culture Collection. In RPMI media, the cells were cultured supplemented with 10% FBS and penicillin/streptomycin (100 U/mL). A cell culture incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% humidified air was used to maintain the cells at 37\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. MTT assay\u003c/h2\u003e\u003cp\u003eThe MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide} was used to observe the drugs effect on the proliferation of A549 cancer cells. Approximately 10,000 cells were added to a 96-well plate. Following a 24-hour incubation period, they were treated with cannabidiol (Epidiolex), bevacizumab (Avastin), and their combination. At the end of the 48 hours of treatment, 0.5% MTT solution was added, and the absorbance was recorded using a spectrophotometer at a 560 nm wavelength.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Flowcytometry analysis\u003c/h2\u003e\u003cp\u003eFlowcytometry (BD FACS Celesta) was used for analyzing cell apoptosis. Cells that had reached 80% confluence were treated with cannabidiol, bevacizumab, and their combination for 48 hours. Subsequently, after centrifugation cells were collected and resuspended in 500\u0026micro;l of binding buffer and allowed to incubate for a duration of 15 minutes at room temperature. Followed by addition of 5 \u0026micro;l each of annexin V-FITC and propidium iodide, the data was subjected to flow cytometry analysis with FACSDiva Software 8.0.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Western blot analysis\u003c/h2\u003e\u003cp\u003eThe effect on the apoptotic genes was investigated using western blotting. Cannabidiol, bevacizumab, and their combination were administered to the A549 cancer cells for a 48-hour period. Following the collection of the cells using RIPA buffer, the total protein concentration was measured via the Pierce Biotechnology BCA Protein Assay Kit. For electrophoresis, 50 \u0026micro;g of proteins were run through a 10% SDS-PAGE. The western blot was performed using primary antibodies, caspase-9, Bcl-2, GAPDH and secondary antibodies, IgG-horseradish peroxidase. Elecsys-2010 (Roche Professional Diagnostics) was used to collect the data, and ImageJ software 1.52 was used to calculate the optical densities for each band.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Quantitative real-time polymerase chain reaction (qPCR)\u003c/h2\u003e\u003cp\u003eRNA extraction was executed by TRIzol reagent after treating the cells with cannabidiol, bevacizumab, and their combination. About 1 \u0026micro;g of RNA was then reverse transcribed, and the MMP-2 \u0026amp; -9 mRNA levels were determined with the Qiagen SYBR Green PCR Master Mix using qPCR. GAPDH was used as a reference gene for normalization. The primer sequences used for this method are: MMP-2: 5\u0026rsquo;-CAGGCTCTTCTCCTTTCACAAC-3\u0026rsquo; and 5\u0026rsquo;-AAGCCACGGCTTGGTTTTCCTC-3\u0026rsquo;; MMP-9: 5\u0026rsquo;-TGGGCTACGTGACCTATGACAT-3\u0026rsquo; and 5\u0026rsquo;-GCCCAGCCCACCTCCACTCCTC-3\u0026rsquo;; GAPDH: 5\u0026rsquo;-TGACTTCAACAGCGACACCCA-3\u0026rsquo; and 5\u0026rsquo;-CACCCTGTTGCTGTAGCCAAA-3\u0026rsquo;.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e\u003cp\u003eSPSS version 22 software for statistical analysis was utilized to analyze the data. ANOVA and Tukey\u0026rsquo;s post hoc test were used in comparison of the control and treatment groups. P-values below 0.05 were regarded as statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Effect on proliferation of A549 lung cancer cells\u003c/h2\u003e\u003cp\u003eA549 lung cancer cell growth was inhibited by cannabidiol by 8.6%, 15.4%, and 29.2% at 7.5 \u0026micro;M, 15 \u0026micro;M, and 30 \u0026micro;M. A549 lung cancer cell growth was suppressed by bevacizumab by 11.1%, 18.5%, and 33.3% at 4 \u0026micro;M, 8 \u0026micro;M, and 16 \u0026micro;M, respectively. The combination of cannabidiol and bevacizumab inhibited A549 lung cancer cell growth by 22%, 36.8%, and 67.3%, respectively. These findings demonstrated a concentration-dependent decline in A549 lung cancer cell proliferation with a noticeable reduction in cell proliferation when the two drugs were combined together, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Effect on A549 lung cancer cells apoptosis\u003c/h2\u003e\u003cp\u003eFlowcytometry examination revealed single-drug treatment, and the rate of apoptosis was 15.2% with cannabidiol and 21.9% with bevacizumab, respectively. When both medications were used together, the rate of apoptosis rose to 50.4% in comparison to the control group, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. These results suggest that treating lung cancer with a drug combination has significant therapeutic potential.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Effect on apoptotic genes in A549 lung cancer cells\u003c/h2\u003e\u003cp\u003eThe Western blot findings exhibited a comparison of apoptotic genes in cannabidiol and bevacizumab groups; the anti-apoptotic gene (Bcl-2) expression in the group that received both cannabidiol and bevacizumab was significantly lower (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, compared to the cannabidiol and bevacizumab groups, the expression of pro-apoptotic genes (caspase-9) was considerably higher in the cannabidiol\u0026thinsp;+\u0026thinsp;bevacizumab group, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. These findings imply that co-treating A549 lung cancer cells with cannabidiol and bevacizumab increased the cells apoptotic response via both pathways.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Effect on MMP genes in A549 lung cancer cells\u003c/h2\u003e\u003cp\u003eResults of the qPCR demonstrated that cannabidiol and bevacizumab both downregulated MMP-2 and \u0026minus;\u0026thinsp;9 expression in A549 lung cancer cells; however, the combination of the two drugs exhibited a more pronounced reduction than either drug alone (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe treatment of NSCLC has changed significantly during the past ten years. The FDA has approved bevacizumab to treat patients with mid- and advanced-stage NSCLC [8]. Cannabidiol, on the other hand, is not typically used to treat NSCLC patients, but studies have shown that it has anticancer properties in addition to being a cannabinoid and has the ability to cause cancer cells to undergo apoptosis [18]. Therefore, utilizing A549 cells of lung cancer as an in vitro model, the aim of the current research was to examine if the addition of cannabidiol would enhance the anticancer effect of bevacizumab against NSCLC.\u003c/p\u003e\u003cp\u003eUsing the MTT assay, we first looked at how cannabidiol, bevacizumab, and their combination affected the growth of lung cancer cells. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e Our findings demonstrated that the A549 cell growth was inhibited in a concentration-dependent manner, and these results aligned with the research that Kim et al and Wang et al had reported [2,8]. Our findings also revealed that when two drugs were taken together, the inhibition was more noticeable than when they were taken separately.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eApoptosis, commonly referred to as programmed cell death or cell suicide, is a genetically induced process of cell self-destruction. It is important for the growth and control of tissue homeostasis. It occurs when there is an imbalance between anti-apoptotic and pro-apoptotic proteins [19]. Therefore, we evaluated changes in pro-apoptotic proteins (caspase-9) \u0026amp; anti-apoptotic proteins (Bcl-2) in cannabidiol, bevacizumab, and cannabidiol\u0026thinsp;+\u0026thinsp;bevacizumab-treated A549 cells. Our results revealed that, in contrast to the single-drug approach, the group receiving cannabidiol\u0026thinsp;+\u0026thinsp;bevacizumab had significantly lower Bcl-2 expression and higher caspase-9 expression than the other groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) [2,8,9,20]. These findings confirm that the cannabidiol and bevacizumab combination induces apoptosis in A549 lung cancer cells.\u003c/p\u003e\u003cp\u003eLast but not least, we examined the effects of cannabidiol and bevacizumab on MMP-2 and \u0026minus;\u0026thinsp;9, which are crucial for tumor growth and invasion and are expressed in various human cancers [16,21]. According to our findings, cannabidiol and bevacizumab both individually decreased the MMP-2 and \u0026minus;\u0026thinsp;9 expression levels. These results are consistent with other research that has been published [9,16]. Compared to the single-drug treatment, there was a greater suppression of MMP-2 and MMP-9 expression when cannabidiol and bevacizumab were used in combination.\u003c/p\u003e\u003cp\u003eDue to a lack of funds, we used only one non-small cell lung cancer cell line (A549 cells) for evaluating the effect of the drugs and their underlying molecular mechanism.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study shows that co-treatment of cannabidiol and bevacizumab has a more prominent effect on the growth inhibition of NSCLC than bevacizumab alone. However, to establish the combined therapeutic benefits of these two drugs, a thorough clinical trial is necessary.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthical approval:\u003c/h2\u003e\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Ethical approval was obtained from the Institutional review board of Dow University of Health Sceinces, Karachi, Pakistan. (IRB-3274/DUHS/EXEMPTION/2023/460)\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e\u003cp\u003eNot applicable since study utilized cell line.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent to publish:\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData Sharing Statement\u003c/strong\u003e\u003cp\u003eData is not publicly available due to confidentiality but are available upon request to the corresponding author in de-identifiable form.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: Abdul Qadir, Zurqa Khalid; Methodology: Abdul Qadir, Ayesha Kashmala Ghauri; Formal analysis and investigation: Abdul Qadir, Muhammad Hamza Dawood ; Writing - original draft preparation: Abdul Qadir, Zurqa Khalid, Ayesha Kashmala Ghauri, Muhammad Hamza Dawood, Usama Manzoor, Habib Ur Rehman; Writing - review and editing: Abdul Qadir, Zurqa Khalid, Ayesha Kashmala Ghauri, Muhammad Hamza Dawood, Usama Manzoor, Habib Ur Rehman; Resources: Abdul Qadir, Zurqa Khalid; Supervision: Abdul Qadir, Zurqa Khalid.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eNone.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is not publicly available due to confidentiality but are available upon request to the corresponding author in de-identifiable form.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShen J, Su Z. 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Eur Rev Med Pharmacol Sci. 2015 Nov;19(21):4087-97.\u003c/li\u003e\n\u003cli\u003eKim YS, Li F, O'Neill BE, Li Z. Specific binding of modified ZD6474 (Vandetanib) monomer and its dimer with VEGF receptor-2. Bioconjug Chem. 2013 Nov 20;24(11):1937-44.\u003c/li\u003e\n\u003cli\u003eGo YY, Kim SR, Kim DY, Chae SW, Song JJ. Cannabidiol enhances cytotoxicity of anti-cancer drugs in human head and neck squamous cell carcinoma. Sci Rep. 2020 Nov 26;10(1):20622.\u003c/li\u003e\n\u003cli\u003eTamura D, Saito T, Murata K, Kawashima M, Asano R. Celecoxib exerts antitumor effects in canine mammary tumor cells via COX‑2‑independent mechanisms. Int J Oncol. 2015 Mar;46(3):1393 − 404.\u003c/li\u003e\n\u003cli\u003eHuang H, Song J, Liu Z, Pan L, Xu G. Autophagy activation promotes bevacizumab resistance in glioblastoma by suppressing Akt/mTOR signaling pathway. Oncol Lett. 2018;15(2):1487-94.\u003c/li\u003e\n\u003cli\u003eQadir A, Samad DA, Asif M, Ali MM, Zain S. Investigating the effect of vandetanib and celecoxib combination on angiogenesis. J Taibah Univ Med Sc. 2023;18(5):1011-7.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Apoptosis, Bevacizumab, Cannabidiol, MMP-2, MMP-9","lastPublishedDoi":"10.21203/rs.3.rs-7737896/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7737896/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eLung cancer is the leading cause of cancer-related deaths worldwide. Despite advancements in cancer treatment, the survival rate for patients with non-small cell lung cancer (NSCLC) remains around 15%. Therefore, new therapeutic options are needed for treating the patients with NSCLC.\u003c/p\u003e\u003ch2\u003eObjectives\u003c/h2\u003e\u003cp\u003eThe study's objective was to investigate the effect of the cannabidiol and bevacizumab combination against the human NSCLC and its underlying molecular mechanism using A549 cancer cell line as an \u003cem\u003ein vitro\u003c/em\u003e model.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThe A549 cells were cultured and treated with cannabidiol, bevacizumab, and their combination. After incubation, cell growth was evaluated using the MTT assay, and cell apoptosis was determined using flowcytometry. The impact on apoptotic genes was assessed through western blotting, whereas the expression levels of tumor metastasis genes were analyzed using qPCR.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe proliferation of A549 cells was inhibited in a dose-dependent manner by cannabidiol, bevacizumab, and their combination. The apoptotic rate was 14.1% with cannabidiol and 26.5% with bevacizumab; however, when the two drugs were used in combination, the rate of apoptosis was significantly increased by 52.2%, respectively (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The underlying mechanism of apoptosis was further elaborated by western blotting, which displayed that co-treatment downregulated considerably the anti-apoptotic gene Bcl-2, and upregulated the pro-apoptotic gene caspase-9 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Moreover, in comparison to the individual drug, the co-treatment dramatically decreased the expression of MMP-2 and MMP-9 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThe combination of cannabidiol and bevacizumab has a very potent anticancer effect on the growth inhibition of NSCLC than using either drug alone.\u003c/p\u003e","manuscriptTitle":"Exploring the Efficacy of a Novel Cannabidiol and Bevacizumab Combination in Non-Small Cell Lung Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-10 16:12:30","doi":"10.21203/rs.3.rs-7737896/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":"36e0fc53-814e-4426-a3cc-c86bcc4ab1b0","owner":[],"postedDate":"November 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T16:07:37+00:00","versionOfRecord":{"articleIdentity":"rs-7737896","link":"https://doi.org/10.1007/s40199-026-00599-w","journal":{"identity":"daru-journal-of-pharmaceutical-sciences","isVorOnly":false,"title":"DARU Journal of Pharmaceutical Sciences"},"publishedOn":"2026-03-03 15:59:16","publishedOnDateReadable":"March 3rd, 2026"},"versionCreatedAt":"2025-11-10 16:12:30","video":"","vorDoi":"10.1007/s40199-026-00599-w","vorDoiUrl":"https://doi.org/10.1007/s40199-026-00599-w","workflowStages":[]},"version":"v1","identity":"rs-7737896","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7737896","identity":"rs-7737896","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}