Exploring the apoptosis-inducing potential of Cannabis sativa and cannabidiol in pancreatic cancer

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Motadi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7098531/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 17 You are reading this latest preprint version Abstract Pancreatic cancer is deemed one of the most aggressive types of cancer, with a high mortality rate and poor prognosis. Pancreatic cancer is often diagnosed in advanced stages of which the disease is too aggressive to manage, rendering therapeutic interventions ineffective. Cannabis sativa ( C. sativa ) a medicinal plant popularly known for its psycho-altering characteristics is recently being explored for its anti-cancer properties. However, knowledge on its impact on pancreatic cancer is still limited. This study was aimed at exploring the apoptosis-inducing potential of C. sativa and cannabidiol (CBD) in pancreatic cancer cells. Crude C. sativa extracts were obtained by dissolving dried C. sativa leaves in absolute ethanol, CBD was isolated from the crude extract using column chromatography. The cytotoxic effects of the crude extract and CBD on pancreatic cancer cells (Mia-PaCa2) and normal lung cells (MRC-5) were determined using AlamarBlue reagent. Apoptosis was investigated by analysing morphological changes using light microscopy, Annexin V and Hoechst staining. ATP and caspase-3 and − 7 levels were also determined. DNA fragmentation was analysed using agarose gel electrophoresis. The expression of apoptosis related genes was assessed using quantitative polymerase chain reaction (qPCR) following CBD treatment. The results show that both the crude C. sativa and CBD induced substantial cytotoxic effects on Mia-PaCa2 cells but not MRC-5. These treatments caused significant morphological changes on Mia-PaCa2 cells causing the cells to detach, leading to cell death. The cells also stained positive with Annexin V, indicating an induction of apoptosis. Hoescht staining also confirmed nuclear condensation, another indication of apoptosis induction. Crude C. sativa and CBD led to reduced level of ATP as well as caspase-3 and − 7, with the effects more pronounced with the crude extract. DNA fragmentation was also more distinct in cells treated with the crude extract, validating the notion that both crude C. sativa and CBD induced apoptosis in Mia-PaCa2. This was further supported by the upregulation of BAX, BAK-1, and p53 and the downregulation of BCL-2 a indication of the intrinsic apoptotic pathway activation. In conclusion, both crude and CBD have apoptosis-inducing potential in pancreatic cancer cells. Pancreatic cancer C. sativa Cannabidiol (CBD) Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Pancreatic cancer is one of the most aggressive forms of cancer with a high mortality rate. It is ranked 12th most common and the 6th deadliest type of cancer (Ferlay et al., 2024 ). In 2022, 510,992 new cases with 467,409 deaths were reported, showcasing its overwhelming nature. In South Africa, pancreatic cancer is ranked the 9th most common type of cancer, with a mortality rate of approximately 84% (Ferlay et al., 2024 ). Pancreatic cancer is asymptomatic in its early stages, making it difficult to detect. Unfortunately, once symptoms present themselves, pancreatic cancer is in the advanced stages, offering poor prognosis and rendering the available treatments ineffective (Sung et al., 2021 ). In countries such as South Africa, limited resources and a strong dependence on traditional healers presents additional and unique challenges (Edwards and Greeff, 2017 ). This highlights the need to develop therapeutic treatments that would be effective even in advanced disease stages. Apoptosis is a type of cell death that is programmed to occur under controlled conditions and occurs to remove unwanted and damaged cells, which is essential in maintaining homeostasis (Galluzzi et al., 2018 ). Apoptosis has two distinct pathways: the intrinsic and extrinsic apoptotic pathway. The intrinsic pathway is regulated by members of the B-cell lymphoma protein 2 (BCL-2) family. This family contains pro-apoptotic and anti-apoptotic proteins that are carefully balanced to determine a cell's fate (Singh et al., 2021). The primary role of the BLC-2 family of proteins is to regulate the release of cytochrome c by altering the permeability of the mitochondrial membrane (Singh et al., 2021). The p53 gene also regulates this pathway by stimulating pro-apoptotic proteins of the BCL-2 family or by inhibiting apoptosis through inhibition of anti-apoptotic proteins (Vaseva and Moll, 2009 . The anti-apoptotic proteins consist of B cell lymphoma 2 (BCL-2), and B cell lymphoma extra-large (BCL-XL), whereas the pro-apoptotic proteins include B cell lymphoma-10 (BCL-10), BCL-2 associated X protein (BAX), BCL-2 antagonist/killer 1 (BAK-1), BH3 interacting domain death agonist (BID) (Elmore, 2007 ). The extrinsic pathway is initiated through receptor-mediated interactions. During the initiation step, extracellular ligands such as tumour necrosis factor-alpha (TNF-α), Fas ligand (Fas-L), and TNF-related apoptosis-inducing ligand (TRAIL) bind to the “death” domain of transmembrane receptor type 1 TNF receptor (TNFR1), Fas, and TRAIL receptors, respectively (Jan, 2019 ). This binding recruits the adaptor proteins to bind and form death inducing signalling complexes (DISCs). The DISCs formed consist of the death domain, the adaptor molecule, procaspase-8, procaspase-10 and the cellular FLICE inhibitory proteins (c FLIPs) (Bredesen et al., 2006 ). The formation of these complexes results in the auto-catalytic activation of caspase-8. Apoptosis plays a vital role in preventing cancer by removing damaged or mutated cells. This ensures that the mutated cells are eliminated before they can divide uncontrollably, which can lead to the development of cancerous tumours (Pfeffer and Singh, 2018 ). In cancer, however, the intrinsic apoptotic pathway is often suppressed, enabling the cancerous cells to flourish, increasing invasiveness as the tumour grows. Therefore, targeting and stimulating this pathway would help prevent or fight cancer (Pfeffer and Singh, 2018 ). Medicinal plants have been used for centuries for their health benefits, however, most of these medicinal plants have not been validated scientifically to assess their efficacy and safety. Cannabis sativa i s one of the plants that has gained interest in recent years mainly due to its potential anti-cancer activities ( Odieka et al., 2022 ). It encompasses compounds such as phytocannabinoids, terpenes, flavonoids, and alkaloids, of which there are approximately 120 phytocannabinoids, with Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) being the most abundant (Pintori et al., 2023 ). Areas of research focused on the anti-cancer effects of crude C. sativa and CBD are expanding, however, studies on their effects on pancreatic cancer are still limited. This study was therefore aimed at exploring the apoptosis-inducing potential of crude C. sativa and CBD in pancreatic cancer cells (Mia-PaCa2 cell line). Materials and Methods Materials Permission to harvest, process and use C. sativa was granted by the South African Department of Health permit number POS 292/2022/2023. This variety of C. sativa was CBD biased was identified by Ms B Selepe from University of Johannesburg Department of Botany. The following voucher number in line with national cannabis sativa was allocated BLFU MGM 0018. Human cell lines MRC-5, and Mia-PaCa2 were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). Mia-PaCa2 and MRC-5 were derived from human pancreatic cancer cells and fetal lung fibroblast, respectively. Preparation of crude C. sativa extract C. sativa leaves were left to dry at room temperature; once dry, they were ground into a fine powder using an electric blender. Extraction was carried out using absolute ethanol and water in a 2:20 ratio, 1000 mL of this solution was used to reconstitute 100 g of the ground leaves, and this mixture was left overnight. The mixture was filtered and underwent solvent evaporation at 69˚C. Following evaporation, the extract was left to dry at room temperature. Column chromatography was used to isolate and purify CBD. The column was eluted with a 2:8 ratio of ethyl acetate and hexane at a controlled flow rate. Each fraction was analysed using Thin layer chromatography (TLC) to confirm the presence of CBD; 2 mL of ethyl acetate and 8 mL of hexane were used as the mobile phase. Stock solutions of crude C. sativa and isolated CBD were prepared by dissolving 100 mg of each sample in 1 mL of DMSO to make a final concentration of 100 mg/mL. These were stored in the dark at 4˚C. Cell culture Mia-PaCa2 and MRC-5 cells were cultured in T-75 flasks using Dulbecco’s Modified Eagle’s Medium (DMEM) (4500 mg/L glucose, L-glutamine, and sodium pyruvate) (Sigma), supplemented with 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin (10000 Units/mL Penicillin, 10000 µg/mL Streptomycin) (Thermo Fisher Scientific Inc, Waltham, MA, USA). Cells were maintained in a humidified atmosphere of 5% CO 2 at 37°C. Every few days old media was routinely discarded and replaced with fresh media until cells reached approximately 70–80% confluency. Confluent cells were sub-cultured by adding 1 mL of trypsin and placing the flask in the incubator for 5 minutes, 10 mL of fresh media was poured into each flask, and this cell suspension was used to seed cells into 96 well flat bottom plates. This seeding procedure was followed for subsequent experiments. Cell viability assay The cytotoxic effects of both the crude C. sativa and isolated CBD on Mia-PaCa2 and MRC-5 cells were determined using the alamarBlue cell viability assay (Thermo Fisher Scientific Inc, Waltham, MA, USA). After a 24 hour incubation period, the cells seeded in 96 well plates were treated with either C. sativa and isolated CBD, at various concentrations. The following treatment concentrations were prepared from each stock solution: (100, 50, 25, 12.5, 6.25, 3.125 µg/mL). DMSO (0.1%) was used as a negative control, 1% etoposide was used as a positive control, and the treated cells were incubated for an additional 22 hours, thereafter, 10 µL of AlarmaBlue reagent was added to each well. The plates were covered with foil and incubated for 2 hours and the absorbance read on a plate reader at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The cell viability was calculated using the following equation: % Cell viability = \(\:\frac{\text{F}\text{l}\text{u}\text{o}\text{r}\text{e}\text{s}\text{c}\text{e}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d}\:-\:\text{F}\text{l}\text{u}\text{o}\text{r}\text{e}\text{s}\text{c}\text{e}\text{n}\text{e}\text{c}\text{e}\:\text{o}\text{f}\:\text{b}\text{l}\text{a}\text{n}\text{k}}{\text{F}\text{l}\text{u}\text{o}\text{r}\text{e}\text{s}\text{c}\text{e}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{u}\text{n}\text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d}\:-\:\text{F}\text{l}\text{u}\text{o}\text{r}\text{e}\text{s}\text{c}\text{e}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{b}\text{l}\text{a}\text{n}\text{k}}\:\times\:100\) (1) Analysis of cell morphology following treatment Cell morphology was assessed using 3 different techniques. Each technique followed the following protocol: cells were seeded into three different 6-well plates and incubated for 24 hours and afterwards treated with the IC 50 of crude C. sativa and IC 50 of isolated CBD, 0.1% of DMSO, 1% of etoposide and incubated for a further 24 hours. In the first plate, following the incubation period post treatment, cell morphology was analysed using light microscope at 20× magnification. For analysis using Annexin V, post treatment incubation, the cells were harvested, and the pellet was resuspended in 100 µL of 1× binding buffer and 5 µL of Annexin V was added, and the mixture was incubated in the dark for 15 minutes, afterwards the cells were mounted on microscopic slides and analysed using the fluorescence microscope at 20× magnification. In the last 6 well plate Hoechst staining was used to assess if treatment induced nuclear condensation. Following the 24-hour incubation post treatment, the cells were washed with PBS, and 500 µL of 1:2000 diluted Hoechst stain was added and the cells were incubated in the dark for 20 minutes. After the prescribed time, the Hoechst stain was discarded, and the cells were washed 5 times with PBS. The cells were then scrapped off using a cells scraper mounted on microscopic slides and analysed using the fluorescence microscope at 20× magnification. ATP detection ATP levels were measured using the Mitochondrial ToxGlo™ assay kit (Promega, USA) according to the manufacturer's protocol. Briefly, the cells were seeded in 96-well black plates and incubated for 24 hours. The cells were then treated with the IC 50 of crude C. sativa and IC 50 of isolated CBD, 0.1% of DMSO, 1% of etoposide and incubated for an additional 24 hours. Afterwards, 100 µL of the ATP detection reagent was added into each well, and the plate was covered with foil and placed on a shaker for 15 min. The luminescence was measured using a plate reader and ATP levels were recorded as a mean of Relative Light Units (RLU) and the following equation was used: Relative Luminescence Units (RLU) = Luminescence of sample – Luminescence of blank. (2) Caspase 3/7 activity assay The activation of caspase-3 and − 7 was quantified using Caspase-Glo® 3/7 assay kit (Promega, USA) according to the manufacturer's protocol. Briefly, the cells were seeded in black 96-well plates and incubated for 24 hours. After 24 hours the cells were treated with the IC 50 of crude C. sativa and IC 50 of isolated CBD, 0.1% of DMSO, 1% of etoposide and incubated for a further 24 hours. Following treatment, media was replaced with 100 µL of the Caspase-Glo® 3/7 reagent, the plate was immediately covered with foil and placed on a shaker for 30 seconds and incubated at room temperature for 30 minutes. Luminescence was measured using a plate reader and caspase 3/7 levels were then recorded as RLU using Eq. 2 DNA fragmentation analysis by agarose gel electrophoresis DNA fragmentation, a key feature in apoptosis was analysed using agarose gel electrophoresis. Cells were seeded in T-75 flasks and incubated overnight; the cells were treated with either the IC 50 of crude C. sativa and IC 50 of isolated CBD, 0.1% of DMSO or 1% of etoposide and incubated for an additional 24 hours. For the untreated and those treated with 0.1% DMSO, the cells were scrapped off using a cell scrapper and resuspended in 1 mL of PBS. The cell suspensions related to each cell line and different treatments were transferred into 2 mL centrifuge tubes and centrifuged at 1000 xg for 10 minutes. The pellets were resuspended in 200 µL DNA elution buffer, and the DNA was extracted using the Quick-DNA™ Miniprep Plus kit following the manufacturer's protocol. The nanodrop™ 1000 Spectrophotometer was used to quantify the DNA. Prior to electrophoresis, samples were prepared by adding 2 µL of the loading buffer to 10 µL of the extracted DNA. The DNA was analysed on 1% agarose gel electrophoresis containing 1 µL of ethidium bromide, and the gel was imaged using the ChemiDoc imaging system. qPCR: Gene expression cDNA was synthesised using total RNA that was isolated from cultured cells. The real-time quantitative polymerase chain reaction (qPCR) was performed in a 10 µL reaction mixture containing cDNA, SYBR Green, and nuclease-free water and gene-specific primers (specified in Table 1 ). The samples were then placed on the CFX Connect Real-Time system machine, and the running conditions were as follows: initial denaturation at 95 ˚C for 1 min, followed by 40 cycles of denaturation at 95 ˚C for 15 s annealing and extension at 55 ˚C for 10 s. Lastly, the expression levels were calculated relative to the β-Actin housekeeping gene using the delta-delta Ct (2 −ΔΔCt ) method. Table 1 Primer sequences Gene Sequences β-Actin Primer 1: 5’-GTCTTCCCCTCCATCGTG-3’ Primer 2: 5’-GATGCCGTGCTCGATGG-3’ TNF-α Primer 1: 5’-CCGAGGCAGTCAGATCATCTT-3’ Primer 2: 5’-AGCTGCCCCTCAGCTTGA-3’ Fas-L Primer 1: 5’-GGTTCTGGTTGCCTTGGTAGGA-3’ Primer 2: 5’-CTGTGTGCATCTGGCTGGTAGA-3’ Caspase-8 Primer 1: 5’-AGAAGAGGGTCATCCTGGGAGA-3’ Primer 2: 5’-TCAGGACTTCCTTCAAGGCTGC-3’ p53 Primer 1: 5’-GACACGCTTCCCTGGATTG-3’ Primer 2: 5’-GACGCTAGGATCTGACTGC-3’ BAX Primer 1: 5’-AGAGGTCTTTTTCCGAGCGG-3’ Primer 2: 5’-gcagtgagcccagatca-3’ BAK-1 Primer 1: 5’-TTACCGCCATCAGCAGGAACAG-3’ Primer 2: 5’-GGAACTCTGAGTCATAGCGTCG-3’ BCL-2 Primer 1: 5’-GCTATAACTGGAGAGTGCTGAA-3’ Primer 2: 5’-tgttaatatcagtctactttcctctgtg-3’ Results The cytotoxic effects of crude C. sativa and isolated CBD on Mia-PaCa2 and MRC-5 cells The cytotoxic effects of crude C. sativa were evaluated on both Mia-PaCa2 and MRC-5. Crude C. sativa exhibits a dose-dependent cytotoxicity on Mia-PaCa2 cells, with a resulting IC 50 of 35.47 µg/mL (Fig. 1 B): Higher concentrations of the crude C. sativa between 100 and 50 µg/mL, reduced the cell viability to approximately 40% (P ≤ 0.0001), whereas concentrations in the lower range between 6.25 and 3.125 µg/mL displayed the least impact on cell viability. Conversely, crude C. sativa did not cause any notable changes on the cell viability of MRC-5 cells, as seen in Fig. 1 C, this is evidenced by a significantly higher IC 50 of 222.7 µg/mL (Fig. 1 D). Cells treated with 0.1% DMSO showed no significant effect on Mia-PaCa2 and MRC-5 cells, contrarily, 1% etoposide, a known chemotherapeutic agent, displayed significant cytotoxic effects on both Mia-PaCa2 (P ≤ 0.0001) and MRC-5 (P ≤ 0.001) cells. Additional cell viability assays were performed on Mia-PaCa2 and MRC-5 cells using isolated CBD which also showed a dose-dependent cytotoxic effect on Mia-PaCa2 cells (Fig. 2 A ) , similar to crude C. sativa , and an IC 50 of 75.72 µg/mL was obtained (Fig. 2 B). The IC 50 for MRC-5 was 184 µg/mL (Fig. 2 D). Morphological analysis of Mia-PaCa2 cells Assessing the characteristics of apoptosis in Mia-PaCa2 using the light microscope. Morphological changes on Mia-PaCa2 post-treatment were initially analysed using the light microscope. The cells were treated with either 0.1% DMSO, 1% etoposide, IC 50 of crude C. sativa , or the IC 50 of the isolated CBD. The untreated cells (Fig. 3 A) exhibited a spindle or epithelial morphology typical for healthy and proliferative cells. The cells appeared well attached to the plate surface. The cells treated with 0.1% DMSO (Fig. 3 B) showed similar characteristics to untreated Mia-PaCa2 cells. Some DMSO-treated cells acquired a rounder morphology, which is also normal for Mia-PaCa2. In contrast, the cells treated with 1% etoposide, crude C. sativa , and isolated CBD (Fig. 3 C, D, and E ) displayed significant morphological alterations: The cells lost their epithelial morphology and appeared smaller and rounder, consequently detaching from the surface of the plate. Some cells appeared fragmented and showed the presence of apoptotic bodies as highlighted by the white squares in Fig. 3 . Morphological assessment of apoptosis induction using Annexin V staining Annexin V staining was used to confirm the induction of apoptosis by identifying phosphatidylserine exposure on the outer leaflets of the cell membrane. According to Fig. 4 A and B no major differences were observed between untreated and 0.1% DMSO-treated cells showed minimal positive staining with Annexin V. Conversely, the cells treated with 1% etoposide, crude C. sativa and CBD (Fig. 4 C, D and E , respectively) exhibited higher fluorescence intensity. The crude displayed a high percentage of Annexin V-stained cells. Nuclear condensation analysis using Hoechst staining Hoechst staining was used to assess changes in nuclear morphology and visualised using the fluorescence microscope. The nuclei in untreated cells and those treated with 0.1% DMSO as seen in Fig. 5 A and B exhibited brighter fluorescence intensity compared to those treated with 1% etoposide, crude C. sativa , and CBD. These cells also displayed a smooth, evenly stained nuclei. Alternatively, cells treated with either 1% etoposide, crude C. sativa and isolated CBD (Fig. 5 C, D and E ) cells showed reduced fluorescence intensity, and the nuclei of these cells appeared irregular, while some displayed completely dismantled nuclei as highlighted by the white squares. Analysis of mitochondrial ATP and caspase activity following treatment with C. sativa and CBD Analysis of mitochondrial ATP levels and caspase activity were carried out to validate the induction of apoptosis. The untreated cells and those treated with 0.1% DMSO maintained high levels of ATP as observed in Fig. 6 , conversely, those treated with either 1% etoposide, IC 50 crude C. sativa , or IC 50 of isolated CBD exhibited significantly reduced levels of ATP. The Caspase 3/7 assay was used to confirm whether caspases 3 and 7 were involved in the execution of apoptosis. Untreated Mia-PaCa2 cells and those treated with 0.1% DMSO exhibited minimal caspase activity as shown in Fig. 7 , while those treated with 1% etoposide caused an increase in caspase-3 and − 7. Caspase activation was more pronounced in cells treated with the crude C. sativa -treated cells. Caspase-3 and − 7 in CBD-treated cells are comparable to those of etoposide-treated cells. DNA fragmentation and gene expression analysis Apoptosis was further validated by assessing DNA integrity following treatment. Figure 8 shows the DNA of untreated and treated cells. Untreated and 0.1% DMSO-treated cells each displayed a single intact band, with no sign of fragmentation. On the other hand, DNA from cells treated with either 1% etoposide, crude C. sativa or isolated CBD displayed a distinct series of bands consistent with DNA fragmentation. DNA fragmentation was more pronounced in cells treated with crude C. sativa . Gene expression analysis following treatment with CBD To examine the effects of isolated CBD on the expression of genes related to the extrinsic and intrinsic apoptotic pathways, qPCR was used. The expression of key genes TNF-α, FAS-L, and caspase-8, involved in the extrinsic apoptotic pathway following treatment with CBD, which according to Fig. 9 , caused a reduction in the expression of TNF- α, FAS-L, and caspase-8 in Mia-PaCa2. The expression of p53, BAX, BAK-1, and BCL-2; key mediators of the intrinsic apoptotic pathway was analysed following treatment with CBD. CBD upregulated the expression of p53 and the pro-apoptotic genes BAX and BAK-1 but downregulated the expression of the anti-apoptotic gene BCL-2 as seen in Fig. 10 . Discussion Anti-cancer therapeutics that target one or more components of the apoptotic pathways would be a more efficient approach in supressing cancer cell growth and development (Rajabi et al., 2021 ). More and more plant extracts and plant-derived molecules are being explored for their potential as anticancer therapeutics. In their review, Khan et al., 2019 showed that the anticancer properties of secondary metabolites found in plant extracts lies mainly in their ability to cause DNA damage and induce apoptosis in cancer cells. For this study, C. sativa and CBD isolated from the plant extract were used to assess apoptosis induction in Mia-PaCa2 pancreatic cancer cell line. The anti-proliferative effects of Crude C. sativa extracts and isolated CBD were investigated in Mia-PaCa2 and MRC-5 cells, using AlamarBlue assay. According to the guidelines provided by the American National Cancer Institute USA (NCI), plant extracts that have IC 50 values between 20 µg/mL 100 µg/mL are considered moderately active. Both crude C. sativa extracts and isolated CBD were able to reduce the cell viability in Mia-PaCa2 and MRC-5, however, the IC 50 of the crude C. sativa extracts in Mia-PaCa2 showed greater potency compared to the IC 50 obtained from the isolated CBD. The IC 50 values of the crude extract and isolated CBD in MRC-5 are notably higher compared to Mia-PaCa2, which suggests that normal cells would require much higher concentrations to achieve the same level of toxicity. This is a positive result because it implies that if 35.47 µg/mL and 75.72 µg/mL of crude extract and CBD, respectively, are used to treat pancreatic cancer cells, these concentrations will not cause undesired effects on the surrounding normal cells. Further analysis of the IC 50 values shows that in Mia-PaCa2, the crude extract had higher potency compared to CBD, whereas in MRC-5, the opposite is true and CBD exhibited greater cytotoxicity. One possible explanation for increased crude extract potency could be the synergistic effects of the various compounds in crude C. sativa . This phenomenon is referred to as the entourage effect, whereby the different compounds in crude C. sativa work synergistically to enhance the bioactivity of the crude (Surendran et al., 2021 ), making it more effective compared to the isolated CBD. In a study by Blasco-Benito et al. ( 2018 ), crude C. sativa showed enhanced anti-cancer activity compared to THC in various breast cancer cell lines. However, this is not consistent across all cancer types; Lukhele and Motadi ( 2016 ) found that CBD was more effective at inhibiting proliferation of cervical cancer cell lines compared to crude C. sativa . Similarly, Yang et al. ( 2020 ) as well as Mangal et al. ( 2024 ) evaluated the anti-proliferative properties of CBD on various pancreatic cancer cell lines, and they demonstrated that CBD does indeed reduce cell proliferation in a time and dose-dependent manner. Conversly, Romano et al. ( 2014 ) showed no differences in the potency and efficacy of crude C. sativa and CBD in colorectal cancer cell lines. These discrepancies show the complexity of CBD interactions with cancer cells, suggesting that its anti-cancer effects may depend on the cancer type and perhaps the molecular mechanism of CBD and crude in that particular cancer. Cells undergoing apoptosis have specific characteristics which include morphological spikes, blisters, and blebbing (Van Cruchten and Van den Broeck, 2002 ). Following treatment, the morphology of Mia-PaCa2 cells was evaluated using the light microscope, Annexin V and Hoescht stain. Normal Mia-PaCa2 cells exhibit a spindle-shaped appearance and a rounded conformation (Behera et al ., 2021). López-Hernández ( 2021 ) reported that apoptotic cells detach very early from their surrounding tethers and become rounder. Detached cells are more likely to die because they lose access to survival signals provided by adhesion. Under the light microscope, the untreated and those treated with 0.1% DMSO displayed an epithelial morphology which is consistent with normal Mia-PaCa2 cells. On the other hand, the morphological changes observed in 1% etoposide, IC 50 crude C. sativa and CBD-treated cells suggest that the cells were undergoing apoptosis. Annexin V detects early apoptosis by comparing the number of fluorescently stained cells in the treated and control groups. It has a high affinity for phosphatidylserine, a phospholipid enclosed in the inner leaflet of the membrane of healthy cells. This phospholipid gets externalised to the outer leaflet of the membrane during apoptosis, making it easier for Annexin V to bind to it (Rieger et al., 2011 ). Only a few cells were stained positively with Annexin V in the untreated and 0.1% DMSO-treated group, indicating that most cells remained viable. In contrast, the treated cells had a high proportion of cells that stained positive, suggesting they were undergoing apoptosis. These findings align with the previous morphological changes observed using the light microscope. The large number of cells that stained positive for annexin V means that these cells were caught in their early stages of apoptosis. In a similar manner, Choene and Motadi ( 2012 ) as well as Lukhele and Motadi ( 2016 ) indicated that apoptosis was induced after treatment with crude extracts. Hoechst stain was used to assess nuclear condensation, another feature of apoptosis. Hoechst stain is a fluorescent dye that binds to the minor groove of DNA in A-T-rich regions of living cells, blue fluorescent light is emitted after excitation with ultraviolet or blue light (Bucevičius et al., 2018 ). The untreated and those treated with 0.1% DMSO exhibited brighter fluorescence intensity compared to cells treated with etoposide, IC 50 of crude C. sativa , and CBD, indicating intact and uncondensed nuclei morphology. In contrast, the treated cells exhibited reduced fluorescence intensity, and the nuclei of these cells had irregular shapes while some displayed completely dismantled nuclei. Although Hoechst dye binds to DNA, cells undergoing apoptosis have compacted or condensed nuclei, this condensation and possible DNA fragmentation can reduce the amount of DNA available for the dye to bind, which results in reduced fluorescence intensity Crowley et al. ( 2016 ). Toné et al. ( 2007 ) assessed nuclei condensation during apoptosis and identified 3 stages: Stage 1 displays an initial ring condensation, in stage 2 an advanced necklace-like condensation pattern, and in stage 3, complete collapse or disassembly of the nuclei. Based on these findings, most cells treated with etoposide, crude C. sativa , and CBD-treated cells appear to be undergoing stage 2 and stage 3 of nuclei condensation, implying late stage of apoptosis. Apoptosis is also identified according to mitochondrial function as well as caspase 3 and 7 levels. ATP is a marker for cellular energy status; its presence indicates that cells are viable with intact mitochondrial function. The untreated and 0.1% DMSO-treated cells maintained high levels of ATP which shows that cell viability was unaffected. Treated Mia-PaCa2 cells displayed reduced levels of ATP, indicating that the treatments impaired the mitochondrial function and reduced the viability. According to Fiorillo et al. ( 2021 ), high levels of ATP in cancerous cells promotes aggression, the cells exhibit enhanced stem cell-like characteristics, increased migration and spontaneous metastasis. Low levels of ATP therefore suggest that the cells are losing viability, and have impaired mitochondrial function, which often results with cell death. Drummond-Main et al., (2022), showed that CBD lowered the threshold for calcium ions, induced mitochondrial transition pore activation and hindered the uptake of mitochondrial calcium ions, thus leading to mitochondrial dysfunction and decreased ATP production. Conversely, the crude extract showed enhanced ATP level reduction compared to isolated CBD. Once again, this could be due to the ‘entourage effect’, which may amplify the inhibition of ATP production compared to the isolated CBD. Caspase-3 and − 7 are the key executioners of apoptosis and are activated in the later stages, and result in the cleavage of cellular components, DNA fragmentation and other morphological alterations in apoptotic cells (Motadi et al., 2023 ). Low caspase-3 and − 7 levels suggest that apoptosis was not activated, as was the case with untreated cells and those treated with 0.1% DMSO. In contrast, cells treated with 1% etoposide, crude C. sativa, isolated CBD showed increased caspase-3 and − 7 levels. This increased expression suggests activation of caspase-3 and − 7, confirming the initiation of the executioner apoptosis pathway. In fact, Motadi et al. ( 2023 ) reported that the anti-proliferative effects of C. sativa and CBD were due to caspase-3 and − 7 activation. DNA fragmentation is another hallmark of apoptosis, characterised by the cleavage of DNA into oligonucleosomal fragments. Activated caspases mediate this process; activated caspase-3 and − 7 cleave proteins such as poly ADP-ribose polymerase which are responsible for maintaining the integrity of DNA, leaving DNA vulnerable to fragmentation (Yan et al., 2006 ; Agarwal et al., 2009 ). The untreated and 0.1% DMSO-treated cells displayed intact DNA with no signs of fragmentation. Alternatively, cells treated with etoposide, crude C. sativa , and isolated CBD-treated cells displayed a distinct series of bands, indicating apoptotic cell death. Apoptosis can be classified through the extrinsic or intrinsic pathways. To determine which of these pathways was activated, CBD was used exclusively to treat Mia-PaCa2 cells as it would provide better elucidation of the molecular pathway: C. sativa has too many compounds and following the metabolic pathways responsible for activating apoptosis would be challenging to follow at this stage. To determine whether CBD mediated cell death through the extrinsic or intrinsic apoptotic pathway the expression of apoptosis-related genes was analysed using qPCR. TNF- α is a cytokine that plays a role in stimulating inflammation by triggering an immune response. It induces apoptosis by activating the extrinsic pathway (van Loo and Bertrand, 2022). It is often elevated in pancreatic cancer, contributing to tumour progression and metastasis (Gao et al., 2023 ). On the other hand, FAS is a death cell surface receptor, and its activation also triggers the extrinsic apoptotic pathway (Kaufmann et al ., 2011). FAS expression in pancreatic cancer is aberrant; however, Kornmann et al. ( 2000 ) reported that although pancreatic cancer exhibited high FAS expression, it is also resistant to FAS-mediated apoptosis. Caspase-8 is an initiator in the extrinsic pathway, and it is usually activated through the activation of FAS, leading to the formation of DICS. Once activated, it activates the executioner caspases (Tummers and Green, 2017 ). Jamil et al. ( 2015 ) reported that although pancreatic cancer cells show high expression of caspase-8, they still failed to trigger apoptosis, suggesting that the activation of caspase-8 alone is insufficient to induce apoptosis. The expressions of TNF-α, Fas-L and caspase-8 genes in MiaPaC-2 were significantly downregulated by treatment with CBD. These findings are consistent with Emhemmed et al. ( 2022 ), who reported that CBD inhibited the secretion of TNF- α. By reducing the expression of TNF- α, CBD can mitigate the pro-inflammatory environment that supports tumour progression and metastasis. Low expression of FAS implies that CBD does not induce FAS-mediated apoptosis. Furthermore, low FAS gene expression levels will inherently mean that caspase 8 expression will also be low. The reduced the expressions of TNF-α, Fas-L, and caspase-8 indicates that the extrinsic apoptotic pathway was not activated by CBD. The expression levels of dominant genes involved in the intrinsic pathway were analysed and results show that CBD caused p53, BAX, and BAK-1 to be upregulated and BCL-2 downregulated, suggesting a coordinated shift towards cell death. p53 is considered ‘the guardian of the genome’ and is critical in regulating apoptosis in response to cellular stress (Hernández Borrero and El-Deiry, 2021 ). By upregulating p53, CBD may enhance the cell's apoptotic response to stress, this is further supported by increased expression of BAX and BAK-1 since they promote mitochondrial outer membrane permeabilization (MOMP), a critical step in the intrinsic apoptotic pathway. Once MOMP occurs, cytochrome c is released from the mitochondrial which in turn causes the activation of executioners' caspases (Jan and Chaudhry, 2019; Tshabalala et al, 2025 ). The upregulation of the pro-apoptotic genes suggests increased apoptotic signals since caspase-3 and − 7 were also activated. Although there is limited evidence on how CBD regulates the expression of BAX and BAK-1 in pancreatic cancer cells, it still appears to be a common occurrence as Lukhele and Motadi ( 2016 ) reported that CBD increased the expression of BAX in cervical cancer. Similarly, Shrivastava et al. ( 2011 ) also reported that CBD increased the expression of BAX, but the expression of BAK-1 remained unchanged. The main function of BCL-2 is to inhibit MOMP (Hardwick and Soane, 2013 ), therefore, reduced BCL-2 expression due to CBD means that BCL-2 is unable to hinder MOMP, thus making cells more susceptible to apoptosis. Proposed mechanism Based on these findings, we propose that CBD induces apoptosis through the intrinsic pathway by acting as an external stressor, activating p53, which in turn leads to the transcription of BCL-2 family of pro-apototic genes, BAX and BAK-1. Activated BAX and BAK-1 induce MOMP and the permeability of outer mitochondria membrane MOMP is considered irreversible as cell death inducing proteins such as cytochrome c are released into the cytosol. Cytochrome c triggers the activation of executioner caspases 3 and − 7, leading to apoptosis. This is depicted in Fig. 11 . Conclusion This study was aimed at exploring the apoptosis-inducing potential of crude C. sativa and CBD in pancreatic cancer cells, thus restoring the cell's apoptotic machinery. The results demonstrated that crude C. sativa and CBD promoted apoptosis by inducing cell detachment and shrinkage, externalising phosphatidylserine, condensing the nucleus, reducing levels of ATP, activating executioner caspases, and DNA fragmentation, all of which are the hallmarks of apoptosis. Furthermore, analysis of gene reveals that CDB increased the expression of pro-apoptotic genes and reduced the expression of anti-apoptotic genes involved in the mitochondrial mediated apoptotic pathway. These results suggest that CBD might inhibit cancer by activating intrinsic apoptotic pathway. However, this needs to be validated by further in in vivo studies. Declarations Conflicts of Interest The Authors declare no conflict of interest in relation to this study. Clinical Trial statement There is no clinical trials at this stage. Ethics approval and consent to participate There are no ethics implications in the study. Consent for publication All authors consent to the work being published in the BMC publication. Availability of data and materials Additional data will be available on request Competing Interests There are no competing interest from all authors mentioned Funding The work was funded by SAMRC and URC-UJ Authors' contributions All authors contributed equally in the preparations and conceptualization of the manuscript. Acknowledgements Authors would like to acknowledge Ms Dimpho Tshabalala and Ms Bridget Lebohang Selepe for the identification of the plant. Abbreviations There are no special abbreviations that require mentioning all are universal References Ferlay, J., Laversanne, M., Ervik, M., Lam, F., Colombet, M., Mery, L., Piñeros, M., Znaor, A., Soerjomataram, I.B.F. and Bray, F., 2024. Global Cancer Observatory: Cancer Tomorrow (version 1.1). Lyon, France: International Agency for Research on Cancer Available from: https://gco. iarc. fr/tomorrow. Accessed , 30 . 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Fas death receptor signalling: roles of Bid and XIAP. Cell Death & Differentiation , 19 (1), pp.42-50. Kornmann, M., Ishiwata, T., Kleeff, J., Beger, H.G. and Korc, M., 2000. Fas and Fas-ligand expression in human pancreatic cancer. Annals of surgery , 231 (3), pp.368-379. Tummers, B. and Green, D.R., 2017. Caspase‐8: regulating life and death. Immunological reviews , 277 (1), pp.76-89. Jamil, S., Lam, I., Majd, M., Tsai, S.H. and Duronio, V., 2015. Etoposide induces cell death via mitochondrial-dependent actions of p53. Cancer Cell International , 15 , pp.1-11. Borrero, L.J.H. and El-Deiry, W.S., 2021. Tumor suppressor p53: Biology, signaling pathways, and therapeutic targeting. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer , 1876 (1), p.188556. Shrivastava, A., Kuzontkoski, P.M., Groopman, J.E. and Prasad, A., 2011. Cannabidiol induces programmed cell death in breast cancer cells by coordinating the cross-talk between apoptosis and autophagy. 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Supplementary Files supplement.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 30 Sep, 2025 Reviews received at journal 28 Sep, 2025 Reviews received at journal 24 Sep, 2025 Reviews received at journal 23 Sep, 2025 Reviews received at journal 15 Sep, 2025 Reviews received at journal 09 Sep, 2025 Reviewers agreed at journal 07 Sep, 2025 Reviewers agreed at journal 03 Sep, 2025 Reviews received at journal 01 Sep, 2025 Reviewers agreed at journal 31 Aug, 2025 Reviewers agreed at journal 29 Aug, 2025 Reviewers agreed at journal 29 Aug, 2025 Reviewers agreed at journal 29 Aug, 2025 Reviewers invited by journal 29 Aug, 2025 Editor assigned by journal 24 Aug, 2025 Submission checks completed at journal 19 Aug, 2025 First submitted to journal 19 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-7098531","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":509530615,"identity":"f8e68db6-2c38-4609-a6db-5bb92fbb7530","order_by":0,"name":"Xolisiwe Sebutsoe","email":"","orcid":"","institution":"University of Johannesburg","correspondingAuthor":false,"prefix":"","firstName":"Xolisiwe","middleName":"","lastName":"Sebutsoe","suffix":""},{"id":509530616,"identity":"798b8a18-dd31-4f5a-96bd-90daa64e6255","order_by":1,"name":"Lesetja R. Motadi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACPjDJJiHHwHCASC1sDAyMDUAtxiRrYUhsINphbGKHjz/4UWaRvuHgAcYPPxjq5AlrkU5LbOw5J5G74cABZskeBjZDgtaxSecYNvC2gbUwSDMw8DASpaXxb5tEugHQlt8MDBL2RGlpBtqSANTCBrTFgHA4gPwyW+achOHMAwfbLHsMEpIJauGXTj7w8U1ZnTzfjcOHb/yoqLMlqAUBJA4CFRsQrx5kHwnGj4JRMApGwcgCAPAYOSbiXBC3AAAAAElFTkSuQmCC","orcid":"","institution":"University of the Johannesburg","correspondingAuthor":true,"prefix":"","firstName":"Lesetja","middleName":"R.","lastName":"Motadi","suffix":""}],"badges":[],"createdAt":"2025-07-11 06:53:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7098531/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7098531/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90665110,"identity":"4e4bc10f-e38e-46c3-ac74-175d9bd50a3b","added_by":"auto","created_at":"2025-09-05 12:24:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":46408,"visible":true,"origin":"","legend":"\u003cp\u003eThe cytotoxic effects of crude \u003cem\u003eC. sativa\u003c/em\u003e on Mia-PaCa2 and MRC-5 cell lines. The bar graphs represent the cell viability percentages for serially diluted crude \u003cem\u003eC. sativa\u003c/em\u003e along with the control groups (The untreated, 0.1% DMSO, and 1% etoposide) for each cell line. Data was expressed as mean ± SD, ns represents p \u0026gt; 0.05. * Represents P ≤ 0.05, ** represents P ≤ 0.01, *** represents P ≤ 0.001, and **** represents P ≤ 0.0001. The IC\u003csub\u003e50\u003c/sub\u003e was deduced using GraphPad Prism 8.4.3. 686 (x64).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7098531/v1/784bc64851bc873473689ba6.png"},{"id":90664256,"identity":"19c7cdc4-91eb-404a-8ccb-cec17c8fc971","added_by":"auto","created_at":"2025-09-05 12:16:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":46330,"visible":true,"origin":"","legend":"\u003cp\u003eThe cytotoxic effects of the isolated CBD on Mia-PaCa2 and MRC-5. The bar graphs represent the cell viability percentages for serially diluted CBD along with the control groups (The untreated, 0.1% DMSO, and 1% etoposide) and a dose-dependent graph for each cell line. Data was expressed as mean ± SD, ns represents p \u0026gt; 0.05. * Represents P ≤ 0.05, ** represents P ≤ 0.01, *** represents P ≤ 0.001, and **** represents P ≤ 0.0001. The IC\u003csub\u003e50\u003c/sub\u003e was deduced using GraphPad Prism 8.4.3. 686 (x64).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7098531/v1/417e672223d3b0fefc6ddbf5.png"},{"id":90665111,"identity":"41d1caae-852b-407e-aded-53e130afb693","added_by":"auto","created_at":"2025-09-05 12:24:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":474188,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological analysis of Mia-PaCa2 cells following treatment. The cells were either untreated (A) or were treated with 0.1% DMSO (B), 1% etoposide (C), and the IC\u003csub\u003e50\u003c/sub\u003e of crude \u003cem\u003eC. sativa\u003c/em\u003e (35.47 µg/mL) (D) and isolated CBD (75.72 µg/mL) (E), incubated for an additional 24 hours. The cells were viewed under the light microscope using 20× magnification. The white blocks indicate apoptotic bodies.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7098531/v1/5f79a5dc9581f709c0e41335.png"},{"id":90665114,"identity":"eb169fa2-0be9-47dd-bf45-19854c5b46ed","added_by":"auto","created_at":"2025-09-05 12:24:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":146466,"visible":true,"origin":"","legend":"\u003cp\u003ePhosphatidylserine externalisation analysis of Mia-PaCa2 cells following treatment.\u0026nbsp; The cells were either untreated (\u003cstrong\u003eA\u003c/strong\u003e) or treated with 0.1% DMSO (B), 1% etoposide (C), and the IC\u003csub\u003e50\u003c/sub\u003e of crude \u003cem\u003eC. sativa\u003c/em\u003e (35.47 µg/mL) (\u003cstrong\u003eD\u003c/strong\u003e) and isolated CBD (75.72 µg/mL) (\u003cstrong\u003eE\u003c/strong\u003e), incubated for an additional 24 hours. The cells were harvested, stained with Annexin V, mounted on microscopic slides, and viewed using a fluorescence microscope under 20× magnification.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7098531/v1/6002b29d58004e4d5d09d28c.png"},{"id":90664257,"identity":"b0ec871a-ae7f-4e3c-8381-32f8d7fc55f1","added_by":"auto","created_at":"2025-09-05 12:16:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":122098,"visible":true,"origin":"","legend":"\u003cp\u003eNuclear condensation analysis of Mia-PaCa2 cells showing untreated (A) or those treated with 0.1% DMSO (B), 1% etoposide (C), and the IC\u003csub\u003e50\u003c/sub\u003e of crude \u003cem\u003eC. sativa\u003c/em\u003e (35.47 µg/mL) (D) and isolated CBD (75.72 µg/mL) (\u003cstrong\u003eE\u003c/strong\u003e), incubated for an additional 24 hours. The cells were harvested, stained with Hoechst stain, mounted on microscopic slides, and viewed using a fluorescence microscope under 20× magnification. The white squares highlight the condensed nucleus.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7098531/v1/d15e4b1927a804bc3a1a4b67.png"},{"id":90665113,"identity":"f5bd8107-34c6-4148-8a3f-6b587ca63ad8","added_by":"auto","created_at":"2025-09-05 12:24:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":15493,"visible":true,"origin":"","legend":"\u003cp\u003eATP levels in Mia-PaCa2 cells following treatment. The cells were treated with 0.1% DMSO, 1% etoposide, and the IC\u003csub\u003e50\u003c/sub\u003e of crude \u003cem\u003eC. sativa \u003c/em\u003e(35.47 µg/mL) and isolated CBD (75.72 µg/mL), incubated for an additional 24 hours. Following treatment, the ATP detection solution was added, and luminescence was measured. Data was expressed as mean ± SD, and the statistical significance was evaluated using student's T-Test where ns represnts that p \u0026gt; 0.05. * Represents P ≤ 0.05, ** represents P ≤ 0.01, *** represents P ≤ 0.001, and **** represents P ≤ 0.0001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7098531/v1/fa9b43572ba8b7508b6010b0.png"},{"id":90664260,"identity":"f00cf3f2-0f55-4691-b14d-b3aac76f76f1","added_by":"auto","created_at":"2025-09-05 12:16:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10491,"visible":true,"origin":"","legend":"\u003cp\u003eCaspase-3 and -7 levels in Mia-PaCa2 cells. The cells were treated with 0.1% DMSO, 1% etoposide, and the IC\u003csub\u003e50\u003c/sub\u003e of crude \u003cem\u003eC. sativa \u003c/em\u003e(35.47 µg/mL) and isolated CBD (75.72 µg/mL) and incubated for 24 h. Following treatment, the capspase3/7 Glo substrate solution was added, and luminescence was measured. Data was expressed as mean ± SD, and the statistical significance was evaluated using student's T-Test where ns represents p \u0026gt; 0.05. * Represents P ≤ 0.05, ** represents P ≤ 0.01, *** represents P ≤ 0.001, and **** represents P ≤ 0.0001.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7098531/v1/6c1586d8586f1db674430849.png"},{"id":90664270,"identity":"36485fe3-a1c6-448d-a033-488f107332b9","added_by":"auto","created_at":"2025-09-05 12:16:28","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":135447,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of DNA Fragmentation in Mia-PaCa2 cells. The cells were treated with 0.1% DMSO, 1% etoposide, and the IC\u003csub\u003e50\u003c/sub\u003e of crude \u003cem\u003eC. sativa \u003c/em\u003e(35.47 µg/mL) and isolated CBD (75.72 µg/mL), incubated for a further 24 hours. DNA was extracted and analysed on 1% agarose gel electrophoresis at 80 voltage for 40 minutes. The fragments were visualised using ethidium bromide under UV light. Lane M represents the DNA marker.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7098531/v1/4143b75af7f7be1217aade55.png"},{"id":90665365,"identity":"70fff261-cb8f-4ea7-aab2-00845843200f","added_by":"auto","created_at":"2025-09-05 12:32:28","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":29895,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression analysis of genes involved in the extrinsic pathway. The cells were treated with 0.1% DMSO, 1% etoposide, and the IC\u003csub\u003e50\u003c/sub\u003e of isolated CBD (75.72 µg/mL), incubated for an additional 24 hours. Following treatment, RNA was extracted, and cDNA was generated. TNF- α, FAS-L and caspase-8 expression was analysed using qPCR, and the relative fold expression was calculated using the delta-delta method. β-actin was used as a housekeeping gene. Data was expressed as mean ± SD, and the statistical significance was evaluated using the student's T-Test where ns represents p \u0026gt; 0.05. * Represents P ≤ 0.05, ** represents P ≤ 0.01, *** represents P ≤ 0.001, and **** represents P ≤ 0.0001.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7098531/v1/9db04ffc6ca0ae17a0c961c6.png"},{"id":90664266,"identity":"9f995783-ceb3-4ab8-8f4b-495ddfd71621","added_by":"auto","created_at":"2025-09-05 12:16:28","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":34065,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression analysis of genes involved in the intrinsic pathway. The cells were treated with 0.1% DMSO, 1% etoposide, and the IC\u003csub\u003e50\u003c/sub\u003e of isolated CBD (75.72 µg/mL), incubated for an additional 24 hours. Following treatment, RNA was extracted, and cDNA was generated. p53, BAX, BAK-1, and BCL-2 expression was analysed using qPCR, and the relative fold expression was calculated using the delta-delta method. β-actin was used as a housekeeping gene. Data was expressed as mean ± SD, and the statistical significance was evaluated using the student's T-Test where ns represents p \u0026gt; 0.05. * Represents P ≤ 0.05, ** represents P ≤ 0.01, *** represents P ≤ 0.001, and **** represents P ≤ 0.0001.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-7098531/v1/3176d5fd1d9921d38bf139cb.png"},{"id":90664274,"identity":"44361245-d468-4df8-bc47-9cf79fe87920","added_by":"auto","created_at":"2025-09-05 12:16:28","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":482888,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic showing the proposed mechanism by which CBD indueced apoptosis in Mia-PaCa2 cells. CBD acts as a stressor, activating p53 and causing the transcription of BAX and BAK-1. Once activated BAX and BAK-1 MOMP is induced leading to the release of cytochrome c. Release of cytochrome c activates the executioner caspases 3 and -7, thus inducing apoptosis.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-7098531/v1/2e5f5a2a851e4ab248557e1f.png"},{"id":90666123,"identity":"e3224354-8dc5-49d9-a82e-43b3a706cd20","added_by":"auto","created_at":"2025-09-05 12:40:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2458175,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7098531/v1/e8c8cef1-931d-47fb-a559-58484626f7b3.pdf"},{"id":90664254,"identity":"9f991f6f-1a16-4c51-bf6a-4dd4caae5b22","added_by":"auto","created_at":"2025-09-05 12:16:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":43863,"visible":true,"origin":"","legend":"","description":"","filename":"supplement.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7098531/v1/9e49d897656eee6670df6f90.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring the apoptosis-inducing potential of Cannabis sativa and cannabidiol in pancreatic cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatic cancer is one of the most aggressive forms of cancer with a high mortality rate. It is ranked 12th most common and the 6th deadliest type of cancer (Ferlay et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In 2022, 510,992 new cases with 467,409 deaths were reported, showcasing its overwhelming nature. In South Africa, pancreatic cancer is ranked the 9th most common type of cancer, with a mortality rate of approximately 84% (Ferlay et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePancreatic cancer is asymptomatic in its early stages, making it difficult to detect. Unfortunately, once symptoms present themselves, pancreatic cancer is in the advanced stages, offering poor prognosis and rendering the available treatments ineffective (Sung et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In countries such as South Africa, limited resources and a strong dependence on traditional healers presents additional and unique challenges (Edwards and Greeff, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This highlights the need to develop therapeutic treatments that would be effective even in advanced disease stages.\u003c/p\u003e\u003cp\u003eApoptosis is a type of cell death that is programmed to occur under controlled conditions and occurs to remove unwanted and damaged cells, which is essential in maintaining homeostasis (Galluzzi et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Apoptosis has two distinct pathways: the intrinsic and extrinsic apoptotic pathway. The intrinsic pathway is regulated by members of the B-cell lymphoma protein 2 (BCL-2) family. This family contains pro-apoptotic and anti-apoptotic proteins that are carefully balanced to determine a cell's fate (Singh et al., 2021). The primary role of the BLC-2 family of proteins is to regulate the release of cytochrome c by altering the permeability of the mitochondrial membrane (Singh et al., 2021). The p53 gene also regulates this pathway by stimulating pro-apoptotic proteins of the BCL-2 family or by inhibiting apoptosis through inhibition of anti-apoptotic proteins (Vaseva and Moll, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e. The anti-apoptotic proteins consist of B cell lymphoma 2 (BCL-2), and B cell lymphoma extra-large (BCL-XL), whereas the pro-apoptotic proteins include B cell lymphoma-10 (BCL-10), BCL-2 associated X protein (BAX), BCL-2 antagonist/killer 1 (BAK-1), BH3 interacting domain death agonist (BID) (Elmore, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe extrinsic pathway is initiated through receptor-mediated interactions. During the initiation step, extracellular ligands such as tumour necrosis factor-alpha (TNF-α), Fas ligand (Fas-L), and TNF-related apoptosis-inducing ligand (TRAIL) bind to the \u0026ldquo;death\u0026rdquo; domain of transmembrane receptor type 1 TNF receptor (TNFR1), Fas, and TRAIL receptors, respectively (Jan, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This binding recruits the adaptor proteins to bind and form death inducing signalling complexes (DISCs). The DISCs formed consist of the death domain, the adaptor molecule, procaspase-8, procaspase-10 and the cellular FLICE inhibitory proteins (c FLIPs) (Bredesen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The formation of these complexes results in the auto-catalytic activation of caspase-8.\u003c/p\u003e\u003cp\u003eApoptosis plays a vital role in preventing cancer by removing damaged or mutated cells. This ensures that the mutated cells are eliminated before they can divide uncontrollably, which can lead to the development of cancerous tumours (Pfeffer and Singh, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In cancer, however, the intrinsic apoptotic pathway is often suppressed, enabling the cancerous cells to flourish, increasing invasiveness as the tumour grows. Therefore, targeting and stimulating this pathway would help prevent or fight cancer (Pfeffer and Singh, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMedicinal plants have been used for centuries for their health benefits, however, most of these medicinal plants have not been validated scientifically to assess their efficacy and safety. \u003cem\u003eCannabis sativa i\u003c/em\u003es one of the plants that has gained interest in recent years mainly due to its potential anti-cancer activities \u003cem\u003e(\u003c/em\u003eOdieka et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It encompasses compounds such as phytocannabinoids, terpenes, flavonoids, and alkaloids, of which there are approximately 120 phytocannabinoids, with Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) being the most abundant (Pintori et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Areas of research focused on the anti-cancer effects of crude \u003cem\u003eC. sativa\u003c/em\u003e and CBD are expanding, however, studies on their effects on pancreatic cancer are still limited. This study was therefore aimed at exploring the apoptosis-inducing potential of crude \u003cem\u003eC. sativa\u003c/em\u003e and CBD in pancreatic cancer cells (Mia-PaCa2 cell line).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003ePermission to harvest, process and use \u003cem\u003eC. sativa\u003c/em\u003e was granted by the South African Department of Health permit number POS 292/2022/2023. This variety of \u003cem\u003eC. sativa\u003c/em\u003e was CBD biased was identified by Ms B Selepe from University of Johannesburg Department of Botany. The following voucher number in line with national cannabis sativa was allocated BLFU MGM 0018. Human cell lines MRC-5, and Mia-PaCa2 were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). Mia-PaCa2 and MRC-5 were derived from human pancreatic cancer cells and fetal lung fibroblast, respectively.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreparation of crude\u003c/b\u003e \u003cb\u003eC. sativa\u003c/b\u003e \u003cb\u003eextract\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eC. sativa\u003c/em\u003e leaves were left to dry at room temperature; once dry, they were ground into a fine powder using an electric blender. Extraction was carried out using absolute ethanol and water in a 2:20 ratio, 1000 mL of this solution was used to reconstitute 100 g of the ground leaves, and this mixture was left overnight. The mixture was filtered and underwent solvent evaporation at 69˚C. Following evaporation, the extract was left to dry at room temperature.\u003c/p\u003e\u003cp\u003eColumn chromatography was used to isolate and purify CBD. The column was eluted with a 2:8 ratio of ethyl acetate and hexane at a controlled flow rate. Each fraction was analysed using Thin layer chromatography (TLC) to confirm the presence of CBD; 2 mL of ethyl acetate and 8 mL of hexane were used as the mobile phase. Stock solutions of crude \u003cem\u003eC. sativa\u003c/em\u003e and isolated CBD were prepared by dissolving 100 mg of each sample in 1 mL of DMSO to make a final concentration of 100 mg/mL. These were stored in the dark at 4˚C.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eMia-PaCa2 and MRC-5 cells were cultured in T-75 flasks using Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) (4500 mg/L glucose, L-glutamine, and sodium pyruvate) (Sigma), supplemented with 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin (10000 Units/mL Penicillin, 10000 \u0026micro;g/mL Streptomycin) (Thermo Fisher Scientific Inc, Waltham, MA, USA). Cells were maintained in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Every few days old media was routinely discarded and replaced with fresh media until cells reached approximately 70\u0026ndash;80% confluency. Confluent cells were sub-cultured by adding 1 mL of trypsin and placing the flask in the incubator for 5 minutes, 10 mL of fresh media was poured into each flask, and this cell suspension was used to seed cells into 96 well flat bottom plates. This seeding procedure was followed for subsequent experiments.\u003c/p\u003e\n\u003ch3\u003eCell viability assay\u003c/h3\u003e\n\u003cp\u003eThe cytotoxic effects of both the crude \u003cem\u003eC. sativa\u003c/em\u003e and isolated CBD on Mia-PaCa2 and MRC-5 cells were determined using the alamarBlue cell viability assay (Thermo Fisher Scientific Inc, Waltham, MA, USA). After a 24 hour incubation period, the cells seeded in 96 well plates were treated with either \u003cem\u003eC. sativa\u003c/em\u003e and isolated CBD, at various concentrations. The following treatment concentrations were prepared from each stock solution: (100, 50, 25, 12.5, 6.25, 3.125 \u0026micro;g/mL). DMSO (0.1%) was used as a negative control, 1% etoposide was used as a positive control, and the treated cells were incubated for an additional 22 hours, thereafter, 10 \u0026micro;L of AlarmaBlue reagent was added to each well. The plates were covered with foil and incubated for 2 hours and the absorbance read on a plate reader at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The cell viability was calculated using the following equation:\u003c/p\u003e\u003cp\u003e% Cell viability = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{F}\\text{l}\\text{u}\\text{o}\\text{r}\\text{e}\\text{s}\\text{c}\\text{e}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{e}\\text{d}\\:-\\:\\text{F}\\text{l}\\text{u}\\text{o}\\text{r}\\text{e}\\text{s}\\text{c}\\text{e}\\text{n}\\text{e}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{b}\\text{l}\\text{a}\\text{n}\\text{k}}{\\text{F}\\text{l}\\text{u}\\text{o}\\text{r}\\text{e}\\text{s}\\text{c}\\text{e}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{u}\\text{n}\\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{e}\\text{d}\\:-\\:\\text{F}\\text{l}\\text{u}\\text{o}\\text{r}\\text{e}\\text{s}\\text{c}\\text{e}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{b}\\text{l}\\text{a}\\text{n}\\text{k}}\\:\\times\\:100\\)\u003c/span\u003e\u003c/span\u003e (1)\u003c/p\u003e\n\u003ch3\u003eAnalysis of cell morphology following treatment\u003c/h3\u003e\n\u003cp\u003eCell morphology was assessed using 3 different techniques. Each technique followed the following protocol: cells were seeded into three different 6-well plates and incubated for 24 hours and afterwards treated with the IC\u003csub\u003e50\u003c/sub\u003e of crude \u003cem\u003eC. sativa\u003c/em\u003e and IC\u003csub\u003e50\u003c/sub\u003e of isolated CBD, 0.1% of DMSO, 1% of etoposide and incubated for a further 24 hours. In the first plate, following the incubation period post treatment, cell morphology was analysed using light microscope at 20\u0026times; magnification. For analysis using Annexin V, post treatment incubation, the cells were harvested, and the pellet was resuspended in 100 \u0026micro;L of 1\u0026times; binding buffer and 5 \u0026micro;L of Annexin V was added, and the mixture was incubated in the dark for 15 minutes, afterwards the cells were mounted on microscopic slides and analysed using the fluorescence microscope at 20\u0026times; magnification. In the last 6 well plate Hoechst staining was used to assess if treatment induced nuclear condensation. Following the 24-hour incubation post treatment, the cells were washed with PBS, and 500 \u0026micro;L of 1:2000 diluted Hoechst stain was added and the cells were incubated in the dark for 20 minutes. After the prescribed time, the Hoechst stain was discarded, and the cells were washed 5 times with PBS. The cells were then scrapped off using a cells scraper mounted on microscopic slides and analysed using the fluorescence microscope at 20\u0026times; magnification.\u003c/p\u003e\n\u003ch3\u003eATP detection\u003c/h3\u003e\n\u003cp\u003eATP levels were measured using the Mitochondrial ToxGlo\u0026trade; assay kit (Promega, USA) according to the manufacturer's protocol. Briefly, the cells were seeded in 96-well black plates and incubated for 24 hours. The cells were then treated with the IC\u003csub\u003e50\u003c/sub\u003e of crude \u003cem\u003eC. sativa\u003c/em\u003e and IC\u003csub\u003e50\u003c/sub\u003e of isolated CBD, 0.1% of DMSO, 1% of etoposide and incubated for an additional 24 hours. Afterwards, 100 \u0026micro;L of the ATP detection reagent was added into each well, and the plate was covered with foil and placed on a shaker for 15 min. The luminescence was measured using a plate reader and ATP levels were recorded as a mean of Relative Light Units (RLU) and the following equation was used:\u003c/p\u003e\u003cp\u003eRelative Luminescence Units (RLU)\u0026thinsp;=\u0026thinsp;Luminescence of sample \u0026ndash; Luminescence of blank. (2)\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCaspase 3/7 activity assay\u003c/h2\u003e\u003cp\u003eThe activation of caspase-3 and \u0026minus;\u0026thinsp;7 was quantified using Caspase-Glo\u0026reg; 3/7 assay kit (Promega, USA) according to the manufacturer's protocol. Briefly, the cells were seeded in black 96-well plates and incubated for 24 hours. After 24 hours the cells were treated with the IC\u003csub\u003e50\u003c/sub\u003e of crude \u003cem\u003eC. sativa\u003c/em\u003e and IC\u003csub\u003e50\u003c/sub\u003e of isolated CBD, 0.1% of DMSO, 1% of etoposide and incubated for a further 24 hours. Following treatment, media was replaced with 100 \u0026micro;L of the Caspase-Glo\u0026reg; 3/7 reagent, the plate was immediately covered with foil and placed on a shaker for 30 seconds and incubated at room temperature for 30 minutes. Luminescence was measured using a plate reader and caspase 3/7 levels were then recorded as RLU using Eq.\u0026nbsp;2\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDNA fragmentation analysis by agarose gel electrophoresis\u003c/h3\u003e\n\u003cp\u003eDNA fragmentation, a key feature in apoptosis was analysed using agarose gel electrophoresis. Cells were seeded in T-75 flasks and incubated overnight; the cells were treated with either the IC\u003csub\u003e50\u003c/sub\u003e of crude \u003cem\u003eC. sativa\u003c/em\u003e and IC\u003csub\u003e50\u003c/sub\u003e of isolated CBD, 0.1% of DMSO or 1% of etoposide and incubated for an additional 24 hours. For the untreated and those treated with 0.1% DMSO, the cells were scrapped off using a cell scrapper and resuspended in 1 mL of PBS. The cell suspensions related to each cell line and different treatments were transferred into 2 mL centrifuge tubes and centrifuged at 1000 xg for 10 minutes. The pellets were resuspended in 200 \u0026micro;L DNA elution buffer, and the DNA was extracted using the Quick-DNA\u0026trade; Miniprep Plus kit following the manufacturer's protocol. The nanodrop\u0026trade; 1000 Spectrophotometer was used to quantify the DNA. Prior to electrophoresis, samples were prepared by adding 2 \u0026micro;L of the loading buffer to 10 \u0026micro;L of the extracted DNA. The DNA was analysed on 1% agarose gel electrophoresis containing 1 \u0026micro;L of ethidium bromide, and the gel was imaged using the ChemiDoc imaging system.\u003c/p\u003e\n\u003ch3\u003eqPCR: Gene expression\u003c/h3\u003e\n\u003cp\u003ecDNA was synthesised using total RNA that was isolated from cultured cells. The real-time quantitative polymerase chain reaction (qPCR) was performed in a 10 \u0026micro;L reaction mixture containing cDNA, SYBR Green, and nuclease-free water and gene-specific primers (specified in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The samples were then placed on the CFX Connect Real-Time system machine, and the running conditions were as follows: initial denaturation at 95 ˚C for 1 min, followed by 40 cycles of denaturation at 95 ˚C for 15 s annealing and extension at 55 ˚C for 10 s. Lastly, the expression levels were calculated relative to the β-Actin housekeeping gene using the delta-delta Ct (2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e) method.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimer sequences\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSequences\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eβ-Actin\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer 1: 5\u0026rsquo;-GTCTTCCCCTCCATCGTG-3\u0026rsquo;\u003c/p\u003e\u003cp\u003ePrimer 2: 5\u0026rsquo;-GATGCCGTGCTCGATGG-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTNF-α\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer 1: 5\u0026rsquo;-CCGAGGCAGTCAGATCATCTT-3\u0026rsquo;\u003c/p\u003e\u003cp\u003ePrimer 2: 5\u0026rsquo;-AGCTGCCCCTCAGCTTGA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFas-L\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer 1: 5\u0026rsquo;-GGTTCTGGTTGCCTTGGTAGGA-3\u0026rsquo;\u003c/p\u003e\u003cp\u003ePrimer 2: 5\u0026rsquo;-CTGTGTGCATCTGGCTGGTAGA-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eCaspase-8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer 1: 5\u0026rsquo;-AGAAGAGGGTCATCCTGGGAGA-3\u0026rsquo;\u003c/p\u003e\u003cp\u003ePrimer 2: 5\u0026rsquo;-TCAGGACTTCCTTCAAGGCTGC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ep53\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer 1: 5\u0026rsquo;-GACACGCTTCCCTGGATTG-3\u0026rsquo;\u003c/p\u003e\u003cp\u003ePrimer 2: 5\u0026rsquo;-GACGCTAGGATCTGACTGC-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBAX\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer 1: 5\u0026rsquo;-AGAGGTCTTTTTCCGAGCGG-3\u0026rsquo;\u003c/p\u003e\u003cp\u003ePrimer 2: 5\u0026rsquo;-gcagtgagcccagatca-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBAK-1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer 1: 5\u0026rsquo;-TTACCGCCATCAGCAGGAACAG-3\u0026rsquo;\u003c/p\u003e\u003cp\u003ePrimer 2: 5\u0026rsquo;-GGAACTCTGAGTCATAGCGTCG-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBCL-2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer 1: 5\u0026rsquo;-GCTATAACTGGAGAGTGCTGAA-3\u0026rsquo;\u003c/p\u003e\u003cp\u003ePrimer 2: 5\u0026rsquo;-tgttaatatcagtctactttcctctgtg-3\u0026rsquo;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eThe cytotoxic effects of crude C. sativa and isolated CBD on Mia-PaCa2 and MRC-5 cells\u003c/h2\u003e\u003cp\u003eThe cytotoxic effects of crude \u003cem\u003eC. sativa\u003c/em\u003e were evaluated on both Mia-PaCa2 and MRC-5. Crude \u003cem\u003eC. sativa\u003c/em\u003e exhibits a dose-dependent cytotoxicity on Mia-PaCa2 cells, with a resulting IC\u003csub\u003e50\u003c/sub\u003e of 35.47 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB): Higher concentrations of the crude \u003cem\u003eC. sativa\u003c/em\u003e between 100 and 50 \u0026micro;g/mL, reduced the cell viability to approximately 40% (P\u0026thinsp;\u0026le;\u0026thinsp;0.0001), whereas concentrations in the lower range between 6.25 and 3.125 \u0026micro;g/mL displayed the least impact on cell viability. Conversely, crude \u003cem\u003eC. sativa\u003c/em\u003e did not cause any notable changes on the cell viability of MRC-5 cells, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, this is evidenced by a significantly higher IC\u003csub\u003e50\u003c/sub\u003e of 222.7 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Cells treated with 0.1% DMSO showed no significant effect on Mia-PaCa2 and MRC-5 cells, contrarily, 1% etoposide, a known chemotherapeutic agent, displayed significant cytotoxic effects on both Mia-PaCa2 (P\u0026thinsp;\u0026le;\u0026thinsp;0.0001) and MRC-5 (P\u0026thinsp;\u0026le;\u0026thinsp;0.001) cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAdditional cell viability assays were performed on Mia-PaCa2 and MRC-5 cells using isolated CBD which also showed a dose-dependent cytotoxic effect on Mia-PaCa2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, similar to crude \u003cem\u003eC. sativa\u003c/em\u003e, and an IC\u003csub\u003e50\u003c/sub\u003e of 75.72 \u0026micro;g/mL was obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The IC\u003csub\u003e50\u003c/sub\u003e for MRC-5 was 184 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eMorphological analysis of Mia-PaCa2 cells\u003c/h2\u003e\u003cp\u003e\u003cem\u003eAssessing the characteristics of apoptosis in Mia-PaCa2 using the light microscope.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eMorphological changes on Mia-PaCa2 post-treatment were initially analysed using the light microscope. The cells were treated with either 0.1% DMSO, 1% etoposide, IC\u003csub\u003e50\u003c/sub\u003e of crude \u003cem\u003eC. sativa\u003c/em\u003e, or the IC\u003csub\u003e50\u003c/sub\u003e of the isolated CBD. The untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) exhibited a spindle or epithelial morphology typical for healthy and proliferative cells. The cells appeared well attached to the plate surface. The cells treated with 0.1% DMSO (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) showed similar characteristics to untreated Mia-PaCa2 cells. Some DMSO-treated cells acquired a rounder morphology, which is also normal for Mia-PaCa2.\u003c/p\u003e\u003cp\u003eIn contrast, the cells treated with 1% etoposide, crude \u003cem\u003eC. sativa\u003c/em\u003e, and isolated CBD (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D, \u003cb\u003eand E\u003c/b\u003e) displayed significant morphological alterations: The cells lost their epithelial morphology and appeared smaller and rounder, consequently detaching from the surface of the plate. Some cells appeared fragmented and showed the presence of apoptotic bodies as highlighted by the white squares in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMorphological assessment of apoptosis induction using Annexin V staining\u003c/h2\u003e\u003cp\u003eAnnexin V staining was used to confirm the induction of apoptosis by identifying phosphatidylserine exposure on the outer leaflets of the cell membrane. According to Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA \u003cb\u003eand B\u003c/b\u003e no major differences were observed between untreated and 0.1% DMSO-treated cells showed minimal positive staining with Annexin V. Conversely, the cells treated with 1% etoposide, crude \u003cem\u003eC. sativa\u003c/em\u003e and CBD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D \u003cb\u003eand E\u003c/b\u003e, respectively) exhibited higher fluorescence intensity. The crude displayed a high percentage of Annexin V-stained cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eNuclear condensation analysis using Hoechst staining\u003c/h2\u003e\u003cp\u003eHoechst staining was used to assess changes in nuclear morphology and visualised using the fluorescence microscope. The nuclei in untreated cells and those treated with 0.1% DMSO as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA \u003cb\u003eand B\u003c/b\u003e exhibited brighter fluorescence intensity compared to those treated with 1% etoposide, crude \u003cem\u003eC. sativa\u003c/em\u003e, and CBD. These cells also displayed a smooth, evenly stained nuclei. Alternatively, cells treated with either 1% etoposide, crude \u003cem\u003eC. sativa\u003c/em\u003e and isolated CBD (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D \u003cb\u003eand E\u003c/b\u003e) cells showed reduced fluorescence intensity, and the nuclei of these cells appeared irregular, while some displayed completely dismantled nuclei as highlighted by the white squares.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis of mitochondrial ATP and caspase activity following treatment with C. sativa and CBD\u003c/h2\u003e\u003cp\u003eAnalysis of mitochondrial ATP levels and caspase activity were carried out to validate the induction of apoptosis. The untreated cells and those treated with 0.1% DMSO maintained high levels of ATP as observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, conversely, those treated with either 1% etoposide, IC\u003csub\u003e50\u003c/sub\u003e crude \u003cem\u003eC. sativa\u003c/em\u003e, or IC\u003csub\u003e50\u003c/sub\u003e of isolated CBD exhibited significantly reduced levels of ATP.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe Caspase 3/7 assay was used to confirm whether caspases 3 and 7 were involved in the execution of apoptosis. Untreated Mia-PaCa2 cells and those treated with 0.1% DMSO exhibited minimal caspase activity as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, while those treated with 1% etoposide caused an increase in caspase-3 and \u0026minus;\u0026thinsp;7. Caspase activation was more pronounced in cells treated with the crude \u003cem\u003eC. sativa\u003c/em\u003e-treated cells. Caspase-3 and \u0026minus;\u0026thinsp;7 in CBD-treated cells are comparable to those of etoposide-treated cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eDNA fragmentation and gene expression analysis\u003c/h2\u003e\u003cp\u003eApoptosis was further validated by assessing DNA integrity following treatment. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows the DNA of untreated and treated cells. Untreated and 0.1% DMSO-treated cells each displayed a single intact band, with no sign of fragmentation. On the other hand, DNA from cells treated with either 1% etoposide, crude \u003cem\u003eC. sativa\u003c/em\u003e or isolated CBD displayed a distinct series of bands consistent with DNA fragmentation. DNA fragmentation was more pronounced in cells treated with crude \u003cem\u003eC. sativa\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eGene expression analysis following treatment with CBD\u003c/h2\u003e\u003cp\u003eTo examine the effects of isolated CBD on the expression of genes related to the extrinsic and intrinsic apoptotic pathways, qPCR was used. The expression of key genes TNF-α, FAS-L, and caspase-8, involved in the extrinsic apoptotic pathway following treatment with CBD, which according to Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, caused a reduction in the expression of TNF- α, FAS-L, and caspase-8 in Mia-PaCa2.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe expression of p53, BAX, BAK-1, and BCL-2; key mediators of the intrinsic apoptotic pathway was analysed following treatment with CBD. CBD upregulated the expression of p53 and the pro-apoptotic genes BAX and BAK-1 but downregulated the expression of the anti-apoptotic gene BCL-2 as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAnti-cancer therapeutics that target one or more components of the apoptotic pathways would be a more efficient approach in supressing cancer cell growth and development (Rajabi et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). More and more plant extracts and plant-derived molecules are being explored for their potential as anticancer therapeutics. In their review, Khan et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e showed that the anticancer properties of secondary metabolites found in plant extracts lies mainly in their ability to cause DNA damage and induce apoptosis in cancer cells. For this study, \u003cem\u003eC. sativa\u003c/em\u003e and CBD isolated from the plant extract were used to assess apoptosis induction in Mia-PaCa2 pancreatic cancer cell line.\u003c/p\u003e\u003cp\u003eThe anti-proliferative effects of Crude \u003cem\u003eC. sativa\u003c/em\u003e extracts and isolated CBD were investigated in Mia-PaCa2 and MRC-5 cells, using AlamarBlue assay. According to the guidelines provided by the American National Cancer Institute USA (NCI), plant extracts that have IC\u003csub\u003e50\u003c/sub\u003e values between 20 \u0026micro;g/mL 100 \u0026micro;g/mL are considered moderately active. Both crude \u003cem\u003eC. sativa\u003c/em\u003e extracts and isolated CBD were able to reduce the cell viability in Mia-PaCa2 and MRC-5, however, the IC\u003csub\u003e50\u003c/sub\u003e of the crude \u003cem\u003eC. sativa\u003c/em\u003e extracts in Mia-PaCa2 showed greater potency compared to the IC\u003csub\u003e50\u003c/sub\u003e obtained from the isolated CBD. The IC\u003csub\u003e50\u003c/sub\u003e values of the crude extract and isolated CBD in MRC-5 are notably higher compared to Mia-PaCa2, which suggests that normal cells would require much higher concentrations to achieve the same level of toxicity. This is a positive result because it implies that if 35.47 \u0026micro;g/mL and 75.72 \u0026micro;g/mL of crude extract and CBD, respectively, are used to treat pancreatic cancer cells, these concentrations will not cause undesired effects on the surrounding normal cells.\u003c/p\u003e\u003cp\u003eFurther analysis of the IC\u003csub\u003e50\u003c/sub\u003e values shows that in Mia-PaCa2, the crude extract had higher potency compared to CBD, whereas in MRC-5, the opposite is true and CBD exhibited greater cytotoxicity. One possible explanation for increased crude extract potency could be the synergistic effects of the various compounds in crude \u003cem\u003eC. sativa\u003c/em\u003e. This phenomenon is referred to as the entourage effect, whereby the different compounds in crude \u003cem\u003eC. sativa\u003c/em\u003e work synergistically to enhance the bioactivity of the crude (Surendran et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), making it more effective compared to the isolated CBD. In a study by Blasco-Benito et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), crude \u003cem\u003eC. sativa\u003c/em\u003e showed enhanced anti-cancer activity compared to THC in various breast cancer cell lines. However, this is not consistent across all cancer types; Lukhele and Motadi (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) found that CBD was more effective at inhibiting proliferation of cervical cancer cell lines compared to crude \u003cem\u003eC. sativa\u003c/em\u003e. Similarly, Yang et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) as well as Mangal et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) evaluated the anti-proliferative properties of CBD on various pancreatic cancer cell lines, and they demonstrated that CBD does indeed reduce cell proliferation in a time and dose-dependent manner. Conversly, Romano et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) showed no differences in the potency and efficacy of crude \u003cem\u003eC. sativa\u003c/em\u003e and CBD in colorectal cancer cell lines. These discrepancies show the complexity of CBD interactions with cancer cells, suggesting that its anti-cancer effects may depend on the cancer type and perhaps the molecular mechanism of CBD and crude in that particular cancer.\u003c/p\u003e\u003cp\u003eCells undergoing apoptosis have specific characteristics which include morphological spikes, blisters, and blebbing (Van Cruchten and Van den Broeck, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Following treatment, the morphology of Mia-PaCa2 cells was evaluated using the light microscope, Annexin V and Hoescht stain. Normal Mia-PaCa2 cells exhibit a spindle-shaped appearance and a rounded conformation (Behera \u003cem\u003eet al\u003c/em\u003e., 2021). L\u0026oacute;pez-Hern\u0026aacute;ndez (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported that apoptotic cells detach very early from their surrounding tethers and become rounder. Detached cells are more likely to die because they lose access to survival signals provided by adhesion. Under the light microscope, the untreated and those treated with 0.1% DMSO displayed an epithelial morphology which is consistent with normal Mia-PaCa2 cells. On the other hand, the morphological changes observed in 1% etoposide, IC\u003csub\u003e50\u003c/sub\u003e crude \u003cem\u003eC. sativa\u003c/em\u003e and CBD-treated cells suggest that the cells were undergoing apoptosis.\u003c/p\u003e\u003cp\u003eAnnexin V detects early apoptosis by comparing the number of fluorescently stained cells in the treated and control groups. It has a high affinity for phosphatidylserine, a phospholipid enclosed in the inner leaflet of the membrane of healthy cells. This phospholipid gets externalised to the outer leaflet of the membrane during apoptosis, making it easier for Annexin V to bind to it (Rieger et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Only a few cells were stained positively with Annexin V in the untreated and 0.1% DMSO-treated group, indicating that most cells remained viable. In contrast, the treated cells had a high proportion of cells that stained positive, suggesting they were undergoing apoptosis. These findings align with the previous morphological changes observed using the light microscope. The large number of cells that stained positive for annexin V means that these cells were caught in their early stages of apoptosis. In a similar manner, Choene and Motadi (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) as well as Lukhele and Motadi (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) indicated that apoptosis was induced after treatment with crude extracts.\u003c/p\u003e\u003cp\u003eHoechst stain was used to assess nuclear condensation, another feature of apoptosis. Hoechst stain is a fluorescent dye that binds to the minor groove of DNA in A-T-rich regions of living cells, blue fluorescent light is emitted after excitation with ultraviolet or blue light (Bucevičius et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The untreated and those treated with 0.1% DMSO exhibited brighter fluorescence intensity compared to cells treated with etoposide, IC\u003csub\u003e50\u003c/sub\u003e of crude \u003cem\u003eC. sativa\u003c/em\u003e, and CBD, indicating intact and uncondensed nuclei morphology. In contrast, the treated cells exhibited reduced fluorescence intensity, and the nuclei of these cells had irregular shapes while some displayed completely dismantled nuclei. Although Hoechst dye binds to DNA, cells undergoing apoptosis have compacted or condensed nuclei, this condensation and possible DNA fragmentation can reduce the amount of DNA available for the dye to bind, which results in reduced fluorescence intensity Crowley et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Ton\u0026eacute; et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) assessed nuclei condensation during apoptosis and identified 3 stages: Stage 1 displays an initial ring condensation, in stage 2 an advanced necklace-like condensation pattern, and in stage 3, complete collapse or disassembly of the nuclei. Based on these findings, most cells treated with etoposide, crude \u003cem\u003eC. sativa\u003c/em\u003e, and CBD-treated cells appear to be undergoing stage 2 and stage 3 of nuclei condensation, implying late stage of apoptosis.\u003c/p\u003e\u003cp\u003eApoptosis is also identified according to mitochondrial function as well as caspase 3 and 7 levels. ATP is a marker for cellular energy status; its presence indicates that cells are viable with intact mitochondrial function. The untreated and 0.1% DMSO-treated cells maintained high levels of ATP which shows that cell viability was unaffected. Treated Mia-PaCa2 cells displayed reduced levels of ATP, indicating that the treatments impaired the mitochondrial function and reduced the viability. According to Fiorillo et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), high levels of ATP in cancerous cells promotes aggression, the cells exhibit enhanced stem cell-like characteristics, increased migration and spontaneous metastasis. Low levels of ATP therefore suggest that the cells are losing viability, and have impaired mitochondrial function, which often results with cell death. Drummond-Main et al., (2022), showed that CBD lowered the threshold for calcium ions, induced mitochondrial transition pore activation and hindered the uptake of mitochondrial calcium ions, thus leading to mitochondrial dysfunction and decreased ATP production. Conversely, the crude extract showed enhanced ATP level reduction compared to isolated CBD. Once again, this could be due to the \u0026lsquo;entourage effect\u0026rsquo;, which may amplify the inhibition of ATP production compared to the isolated CBD.\u003c/p\u003e\u003cp\u003eCaspase-3 and \u0026minus;\u0026thinsp;7 are the key executioners of apoptosis and are activated in the later stages, and result in the cleavage of cellular components, DNA fragmentation and other morphological alterations in apoptotic cells (Motadi et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Low caspase-3 and \u0026minus;\u0026thinsp;7 levels suggest that apoptosis was not activated, as was the case with untreated cells and those treated with 0.1% DMSO. In contrast, cells treated with 1% etoposide, crude C. sativa, isolated CBD showed increased caspase-3 and \u0026minus;\u0026thinsp;7 levels. This increased expression suggests activation of caspase-3 and \u0026minus;\u0026thinsp;7, confirming the initiation of the executioner apoptosis pathway. In fact, Motadi et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported that the anti-proliferative effects of \u003cem\u003eC. sativa\u003c/em\u003e and CBD were due to caspase-3 and \u0026minus;\u0026thinsp;7 activation.\u003c/p\u003e\u003cp\u003eDNA fragmentation is another hallmark of apoptosis, characterised by the cleavage of DNA into oligonucleosomal fragments. Activated caspases mediate this process; activated caspase-3 and \u0026minus;\u0026thinsp;7 cleave proteins such as poly ADP-ribose polymerase which are responsible for maintaining the integrity of DNA, leaving DNA vulnerable to fragmentation (Yan et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Agarwal et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The untreated and 0.1% DMSO-treated cells displayed intact DNA with no signs of fragmentation. Alternatively, cells treated with etoposide, crude \u003cem\u003eC. sativa\u003c/em\u003e, and isolated CBD-treated cells displayed a distinct series of bands, indicating apoptotic cell death.\u003c/p\u003e\u003cp\u003eApoptosis can be classified through the extrinsic or intrinsic pathways. To determine which of these pathways was activated, CBD was used exclusively to treat Mia-PaCa2 cells as it would provide better elucidation of the molecular pathway: \u003cem\u003eC. sativa\u003c/em\u003e has too many compounds and following the metabolic pathways responsible for activating apoptosis would be challenging to follow at this stage. To determine whether CBD mediated cell death through the extrinsic or intrinsic apoptotic pathway the expression of apoptosis-related genes was analysed using qPCR. TNF- α is a cytokine that plays a role in stimulating inflammation by triggering an immune response. It induces apoptosis by activating the extrinsic pathway (van Loo and Bertrand, 2022). It is often elevated in pancreatic cancer, contributing to tumour progression and metastasis (Gao et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). On the other hand, FAS is a death cell surface receptor, and its activation also triggers the extrinsic apoptotic pathway (Kaufmann \u003cem\u003eet al\u003c/em\u003e., 2011). FAS expression in pancreatic cancer is aberrant; however, Kornmann et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) reported that although pancreatic cancer exhibited high FAS expression, it is also resistant to FAS-mediated apoptosis. Caspase-8 is an initiator in the extrinsic pathway, and it is usually activated through the activation of FAS, leading to the formation of DICS. Once activated, it activates the executioner caspases (Tummers and Green, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Jamil et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) reported that although pancreatic cancer cells show high expression of caspase-8, they still failed to trigger apoptosis, suggesting that the activation of caspase-8 alone is insufficient to induce apoptosis. The expressions of TNF-α, Fas-L and caspase-8 genes in MiaPaC-2 were significantly downregulated by treatment with CBD. These findings are consistent with Emhemmed et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), who reported that CBD inhibited the secretion of TNF- α. By reducing the expression of TNF- α, CBD can mitigate the pro-inflammatory environment that supports tumour progression and metastasis. Low expression of FAS implies that CBD does not induce FAS-mediated apoptosis. Furthermore, low FAS gene expression levels will inherently mean that caspase 8 expression will also be low. The reduced the expressions of TNF-α, Fas-L, and caspase-8 indicates that the extrinsic apoptotic pathway was not activated by CBD.\u003c/p\u003e\u003cp\u003eThe expression levels of dominant genes involved in the intrinsic pathway were analysed and results show that CBD caused p53, BAX, and BAK-1 to be upregulated and BCL-2 downregulated, suggesting a coordinated shift towards cell death. p53 is considered \u0026lsquo;the guardian of the genome\u0026rsquo; and is critical in regulating apoptosis in response to cellular stress (Hern\u0026aacute;ndez Borrero and El-Deiry, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). By upregulating p53, CBD may enhance the cell's apoptotic response to stress, this is further supported by increased expression of BAX and BAK-1 since they promote mitochondrial outer membrane permeabilization (MOMP), a critical step in the intrinsic apoptotic pathway. Once MOMP occurs, cytochrome c is released from the mitochondrial which in turn causes the activation of executioners' caspases (Jan and Chaudhry, 2019; Tshabalala et al, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The upregulation of the pro-apoptotic genes suggests increased apoptotic signals since caspase-3 and \u0026minus;\u0026thinsp;7 were also activated. Although there is limited evidence on how CBD regulates the expression of BAX and BAK-1 in pancreatic cancer cells, it still appears to be a common occurrence as Lukhele and Motadi (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) reported that CBD increased the expression of BAX in cervical cancer. Similarly, Shrivastava et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) also reported that CBD increased the expression of BAX, but the expression of BAK-1 remained unchanged. The main function of BCL-2 is to inhibit MOMP (Hardwick and Soane, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), therefore, reduced BCL-2 expression due to CBD means that BCL-2 is unable to hinder MOMP, thus making cells more susceptible to apoptosis.\u003c/p\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eProposed mechanism\u003c/h2\u003e\u003cp\u003eBased on these findings, we propose that CBD induces apoptosis through the intrinsic pathway by acting as an external stressor, activating p53, which in turn leads to the transcription of BCL-2 family of pro-apototic genes, BAX and BAK-1. Activated BAX and BAK-1 induce MOMP and the permeability of outer mitochondria membrane MOMP is considered irreversible as cell death inducing proteins such as cytochrome c are released into the cytosol. Cytochrome c triggers the activation of executioner caspases 3 and \u0026minus;\u0026thinsp;7, leading to apoptosis. This is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study was aimed at exploring the apoptosis-inducing potential of crude \u003cem\u003eC. sativa\u003c/em\u003e and CBD in pancreatic cancer cells, thus restoring the cell's apoptotic machinery. The results demonstrated that crude \u003cem\u003eC. sativa\u003c/em\u003e and CBD promoted apoptosis by inducing cell detachment and shrinkage, externalising phosphatidylserine, condensing the nucleus, reducing levels of ATP, activating executioner caspases, and DNA fragmentation, all of which are the hallmarks of apoptosis. Furthermore, analysis of gene reveals that CDB increased the expression of pro-apoptotic genes and reduced the expression of anti-apoptotic genes involved in the mitochondrial mediated apoptotic pathway. These results suggest that CBD might inhibit cancer by activating intrinsic apoptotic pathway. However, this needs to be validated by further in \u003cem\u003ein vivo\u003c/em\u003e studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Authors declare no conflict of interest in relation to this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no clinical trials at this stage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no ethics implications in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent to the work being published in the BMC publication.\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional data will be available on request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no competing interest from all authors mentioned\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe work was funded by SAMRC and URC-UJ\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All authors contributed equally in the preparations and conceptualization of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors would like to acknowledge Ms Dimpho Tshabalala and Ms Bridget Lebohang Selepe for the identification of the plant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no special abbreviations that require mentioning all are universal\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eFerlay, J., Laversanne, M., Ervik, M., Lam, F., Colombet, M., Mery, L., Pi\u0026ntilde;eros, M., Znaor, A., Soerjomataram, I.B.F. and Bray, F., 2024. 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Potential biological role of poly (ADP-ribose) polymerase (PARP) in male gametes. \u003cem\u003eReproductive Biology and Endocrinology\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e, pp.1-20.\u003c/li\u003e\n \u003cli\u003eVan Loo, G. and Bertrand, M.J., 2023. Death by TNF: a road to inflammation. \u003cem\u003eNature Reviews Immunology\u003c/em\u003e, \u003cem\u003e23\u003c/em\u003e(5), pp.289-303.\u003c/li\u003e\n \u003cli\u003eGao, Z., Zhang, Q., Chen, H., Chen, J., Kang, J., Yu, H., Song, Y. and Zhang, X., 2023. TNFR2 promotes pancreatic cancer proliferation, migration, and invasion via the NF-\u0026kappa;B signaling pathway. \u003cem\u003eAging (Albany NY)\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(16), p.8013.\u003c/li\u003e\n \u003cli\u003eEmhemmed, F., Zhao, M., Yorulmaz, S., Steyer, D., Leitao, C., Alignan, M., Cerny, M., Paillard, A., Delacourt, F.M., Julien-David, D. and Muller, C.D., 2022. 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Cannabidiol induces programmed cell death in breast cancer cells by coordinating the cross-talk between apoptosis and autophagy. \u003cem\u003eMolecular cancer therapeutics\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(7), pp.1161-1172.\u003c/li\u003e\n \u003cli\u003eHardwick, J.M. and Soane, L., 2013. Multiple functions of BCL-2 family proteins. \u003cem\u003eCold Spring Harbor perspectives in biology\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e(2), p.a008722.\u003c/li\u003e\n \u003cli\u003eChipuk, J.E., Bouchier-Hayes, L. and Green, D.R., 2006. Mitochondrial outer membrane permeabilization during apoptosis: the innocent bystander scenario. \u003cem\u003eCell Death \u0026amp; Differentiation\u003c/em\u003e,\u003cem\u003e13\u003c/em\u003e(8), pp.1396-140\u003c/li\u003e\n \u003cli\u003eTshabalala, D.J., Sebutsoe, X. and Motadi, L., 2025. Exploring the apoptosis-inducing potential of Cannabis sativa and cannabidiol in pancreatic cancer in vitro. \u003cem\u003eCancer Res\u003c/em\u003e, 85 (8_Supplement_1): 1388.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pancreatic cancer, C. sativa, Cannabidiol (CBD), Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-7098531/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7098531/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePancreatic cancer is deemed one of the most aggressive types of cancer, with a high mortality rate and poor prognosis. Pancreatic cancer is often diagnosed in advanced stages of which the disease is too aggressive to manage, rendering therapeutic interventions ineffective. Cannabis sativa (\u003cem\u003eC. sativa\u003c/em\u003e) a medicinal plant popularly known for its psycho-altering characteristics is recently being explored for its anti-cancer properties. However, knowledge on its impact on pancreatic cancer is still limited. This study was aimed at exploring the apoptosis-inducing potential of \u003cem\u003eC. sativa\u003c/em\u003e and cannabidiol (CBD) in pancreatic cancer cells. Crude \u003cem\u003eC. sativa\u003c/em\u003e extracts were obtained by dissolving dried \u003cem\u003eC. sativa\u003c/em\u003e leaves in absolute ethanol, CBD was isolated from the crude extract using column chromatography. The cytotoxic effects of the crude extract and CBD on pancreatic cancer cells (Mia-PaCa2) and normal lung cells (MRC-5) were determined using AlamarBlue reagent. Apoptosis was investigated by analysing morphological changes using light microscopy, Annexin V and Hoechst staining. ATP and caspase-3 and \u0026minus;\u0026thinsp;7 levels were also determined. DNA fragmentation was analysed using agarose gel electrophoresis. The expression of apoptosis related genes was assessed using quantitative polymerase chain reaction (qPCR) following CBD treatment. The results show that both the crude \u003cem\u003eC. sativa\u003c/em\u003e and CBD induced substantial cytotoxic effects on Mia-PaCa2 cells but not MRC-5. These treatments caused significant morphological changes on Mia-PaCa2 cells causing the cells to detach, leading to cell death. The cells also stained positive with Annexin V, indicating an induction of apoptosis. Hoescht staining also confirmed nuclear condensation, another indication of apoptosis induction. Crude \u003cem\u003eC. sativa\u003c/em\u003e and CBD led to reduced level of ATP as well as caspase-3 and \u0026minus;\u0026thinsp;7, with the effects more pronounced with the crude extract. DNA fragmentation was also more distinct in cells treated with the crude extract, validating the notion that both crude \u003cem\u003eC. sativa\u003c/em\u003e and CBD induced apoptosis in Mia-PaCa2. This was further supported by the upregulation of BAX, BAK-1, and p53 and the downregulation of BCL-2 a indication of the intrinsic apoptotic pathway activation. In conclusion, both crude and CBD have apoptosis-inducing potential in pancreatic cancer cells.\u003c/p\u003e","manuscriptTitle":"Exploring the apoptosis-inducing potential of Cannabis sativa and cannabidiol in pancreatic cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-05 12:16:23","doi":"10.21203/rs.3.rs-7098531/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-30T07:19:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-28T19:34:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T08:43:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-23T07:44:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-15T16:27:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-09T12:09:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"146624734567472207524163541180218619655","date":"2025-09-07T20:12:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"96910022303387075508900053431757218892","date":"2025-09-03T08:05:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-01T05:00:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"256550286843824043581881525420653960722","date":"2025-09-01T03:51:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197459339429908011166725443904187708368","date":"2025-08-29T13:29:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"235567073423814345427175366294050842079","date":"2025-08-29T08:16:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"233560318590442716760786564400381860872","date":"2025-08-29T07:28:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-29T06:47:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-24T04:18:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-19T15:49:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Complementary Medicine and Therapies","date":"2025-08-19T15:46:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-complementary-medicine-and-therapies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcam","sideBox":"Learn more about [BMC Complementary Medicine and Therapies](https://bmccomplementmedtherapies.biomedcentral.com/)","snPcode":"","submissionUrl":"","title":"BMC Complementary Medicine and Therapies","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fa5cb353-9ac4-434e-83d3-bf76ebedf843","owner":[],"postedDate":"September 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-10-15T07:08:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-05 12:16:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7098531","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7098531","identity":"rs-7098531","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}