Low dose 5FU incurs DNA repair and induction of EMT in colorectal and pancreatic cancer
An anti-metabolite agent 5FU has been used widely to treat various cancers 24. An anti-metabolite agent 5FU has been used widely to treat various cancers 24. Cancer cells defy the action of DNA damaging drugs like 5FU by inciting epithelial to mesenchymal transition (EMT) challenges the thwarting of apoptotic signals and development of chemoresistance 4, 25. Accordingly, we sought to investigate the effects of 5FU-treatment on DNA damage mediated EMT activation in aggressive cancer cells. The gelatin degradation assay demonstrated significant increase in the matrix degradation activity in 5FU treated HCT-116 cells compared to vehicle (DMSO) treated cells (Fig. 1A).
Since spheroids are excellent model to study neo-vascularization/growth, therefore, we prepared spheroids to study the effects of 5FU on the grown up spheroids. The results showed profound migration of cells from the periphery in 5FU treated spheroids compared to the vehicle wherein such migration from the outer periphery was lacking (Fig. 1B). Additionally the ability of sub-toxic dose 5FU to induce EMT was validated in APC knockout colorectal carcinoma model. After induction of APC floxing, the cohorts were treated with vehicle and 1.6mg/kg 5FU for 48 h. Subsequently, the intestinal tissues obtained were subjected to immunohistochemistry to visualize the expression of vital EMT associated protein Vimentin, which is known to promote invasive phenotype8. Our results showed a substantial Vimentin expression in the 5FU treated group compared to vehicle (Fig. 1C).
Next, to study the effect of 5FU on molecular markers of DDR response mediators and EMT inducer Vimentin, we performed western blotting. The immunoblots depicted steady hike in the expression of DNA damage repair markers viz. XRCC1, RAD51 as well as corresponding upregulation of Vimentin, a structural protein overexpressed in the cells of mesenchymal origin in a similar fashion as positive control etoposide (Fig. 1D). Likewise, our immunocytochemistry results unveiled substantial increase in the expression of Rad51 protein in 5FU treated HCT-116 cells compared to vehicle (Fig. 1E). Moreover, to examine any prominent morphological change in 5FU treated cells, the bright field microscopy images shown a distinct morphological change as demarcated by spindle shaped (EMT phenotype) in HCT-116 and MiaPaca-2 cells following treatment with 5FU (Fig. 1F). Next, to assess the extent of cellular invasion, the transwell Boyden Chamber assay revealed a greater invasive capacity of HCT-116 cells to pierce the transwell membrane following 5FU treatment compared to vehicle treated cells (Fig. 1G). Taken together, the above results demonstrated that 5FU strongly augmented DNA damage mediated EMT activation in in HCT-116 and MiaPaca-2 cells.
THC-9-OG confers antimetastatic property in highly metastatic cancer cells
Recently we have demonstrated o-glycosidic derivative of Δ9-tetrahydrocannabinol as vital EMT modulator in vitro (Patent File No. 0228NF2020) since our Medicinal Chemistry group has isolated distinct C and O-glycosidic Cannabinoids from Cannabis Sativa via divergent synthesis using chemical engineering as a tool. The molecular structure and NMR purity profile of pure compound THC-9-OG is given in Supplementary Fig. 1. Since the anti-metastatic efficacy of psycho-active THC compounds were established in cancer pain management by other groups including us, therefore, in continuation we envisaged the effects of THC-9-OG on DNA damage mediated induction of molecular markers of EMT viz Snail-1, Twist-1, and Vimentin. MIAPaca-2 cells were subjected to immunoblot analysis following treatment with THC-9-OG and the results unveiled significant down-modulation of all the above mentioned EMT markers and simultaneous upregulation of E-cadherin protein in a time and dose-dependent manner (Fig. 2A). Furthermore, to get insight of any morphological alteration of the highly mesenchymal pancreatic cancer cells following THC-9-OG exposure, MIAPaca-2 cells treated with a wide range (0.5-5 µM) of THC-9-OG and were observed under a phase-contrast microscope and the microscopic data depicted significant reversal of mesenchymal morphology of MIAPaca-2 cells by THC-9-OG at 36 h time point (Fig. 2B). Since the basement matrix transformation is a key aspect of metastatic cancer cells, we sought to examine whether THC-9-OG could have any effects on the gelatin matrix in vitro. Hence, the gelatin degradation assay was performed with highly metastatic MIAPaca-2, and MDA-MB-231 cells in presence of THC-9-OG and the results unleashed that 3µM and 5µM concentration of THC-9-OG significantly abrogated invadopodia and filopodia formation, as evident from the significant reduction in percent area of gelatin matrix degradation as compared to vehicle treatment (Fig. 2C). In addition, THC-9-OG also abrogated the invasion capabilities of MIAPaca-2 and MDA-MB-231 cells across the transwell membrane as witnessed by number of less invasive cells compared to vehicle in Boyden chamber assay (Fig. 2D).
To further examine the anti-metastatic effects of THC-9-OG, a breast cancer allograft model was developed by transplanting highly aggressive breast cancer cells (4T1) in a mouse mammary pad of BALB/C mice. The results show that cohorts following tumor development when treated with 15 & 30 mg/kg. of THC-9-OG and 30 mg/kg of DIM (Di-indoyl Methane) (positive control) and normal saline (vehicle) for 15–20 days, a significant reduction of tumor weight and volume by THC-9-OG treatment as compared to vehicle-treated control (Fig. 2E). Moreover, the molecule also inhibited the metastatic spread of 4T1 cells from the breast to the lungs, as evident from the reduction of the number of lung nodules compared to control (Fig. 2E). Next, to study the effect of THC-9-OG on molecular markers of EMT, total proteins isolated from tumors were probed with Snail-1, Twist-1, STAT-3, Vimentin, and E-cadherin antibodies. The immunoblots depicted the abrogation of expression of EMT markers viz Snail-1, Twist-1, Vimentin, and STAT-3 and corresponding upregulation of E-cadherin: a structural protein particular to cells of epithelial origin (Fig. 2F). Cumulatively, these results demonstrate that the glycosidic derivative of tetrahydrocannabinol (THC-9-OG) as a potent antiproliferative and anti-metastatic molecule. Additionally, THC-9-OG is effective in restricting tumor growth and metastasis in 4T1 allograft murine breast cancer model.
THC-9-OG neutralizes 5FU induced EMT in colorectal carcinoma model
Drug resistance is the central issue in cancer chemotherapy affecting its efficacy and, ultimately, patient survival. The data from our previous publication endorsed the idea of using an anti-metastatic molecule to nullify the background EMT/survival effectors associated with DNA damaging drugs to facilitate cell death abrogating the EMT cascade8. To further explore details of drug induced EMT and its abrogation; we choose to study 5FU mediated EMT induction13 and further investigated any effects of THC-9-OG on this forced background EMT pop up. Therefore, gelatin degradation assay was performed in order to assess the effect of THC-9-OG on the 5-FU mediated invasive potential of colon cancer cells. Interestingly, cells treated with 1.5 and 3 µM THC-9-OG combined with 750 nM 5FU displayed reversal of mesenchymal property, resulting in a decrease of gelatin matrix degraded area (Fig. 3A). Furthermore, HCT-116 cells with similar experimental set up as indicated were subjected to immunoblot analysis, and expression of Snail-1, Vimentin, and N-cadherin was examined. The results depicted that the EMT markers, which were upregulated in 750 nM 5FU treated condition, got substantially diminished in THC-9-OG (1.5 & 3 µM) plus 5FU (750 nM) treated lanes (Fig. 3B & C). Further, to validate the abrogation of 5-FU induced acquired migratory property elicited by THC derivative, the 3D spheroid assay was performed. The spheroids prepared were treated with 750 nM 5FU showed profound migration of cells from the periphery compared to the vehicle. Strikingly, the wells treated with 3 µM THC-9-OG combined with 5FU restrained the migration of cells from the spheroid's peripheral boundary (Fig. 3D). The potential of THC-9-OG to abrogate the background EMT activation was additionally validated in APC knockout colorectal carcinoma model. After induction of APC floxing, the cohorts were treated with vehicle, 30 mg/kg THC-9-OG, 1.6mg/kg 5FU, 30 mg/kg THC-9-OG plus 1.6 mg/kg 5FU for 48 h. The intestinal tissues obtained were subjected to immunohistochemistry to visualize the expression of Vimentin. An early appearance of crypt progenitor phenotype in the 5FU-treated mice was found at (1.6 mg/kg) compared to vehicle. The immunohistochemistry analyses of intestinal sections obtained from 5FU-treated mice (1.6 mg/kg) revealed a significant rise in Vimentin expression associated with transformed villi formation whereas THC-9-OG (30 mg/kg) and 5FU (1.6 mg/kg) combination completely diminished Vimentin expression along with restoration of normal villi suggesting the overwhelming efficacy of this combinatorial treatment (Fig. 3E & F). EMT process could potentially aid the cancer cells in evading cell death by promoting anastasis during the initial phases of chemotherapy treatment, consequently leading to the development of secondary tumors that are resistant to chemotherapy26. Further, the intestinal tissues obtained from this Apc flox mice were subjected to prepare lysates and the extracted total protein was analyzed in western blot assay to study the expression of Snail-1 and N-cadherin. The results unveiled the abolition of expression of both the EMT markers in THC-9-OG plus 5FU treated lane as compared to 5FU alone (Fig. 3G & H). Collectively, these results demonstrated that tetrahydrocannabinol derivative (THC-9-OG) abrogated 5-FU induced EMT activation and abrogated crypt progenitor phenotype in vitro and in vivo colorectal carcinoma model.
THC-9-OG alleviates the potency of 5-FU mediated DNA damage
5-Fluorouracil belongs to the DNA damaging agent called antimetabolites, which could be incorporated in place of thymine or uracil, casing activation of DNA damage response 27. The majority of in vitro studies encompass a higher concentration of 5Fluorouracil (> 5 µM) to achieve a significant amount of DNA damage leading to apoptosis 27, 28, 29 .However, in this work we sought to understand the correlation between the anti-metastatic effect of THC-9-OG on the DNA damaging response events of 5-FU when administered at sub-toxic doses. In order to do this, we treated HCT-116 and MiaPaca-2 cells with 5-FU/ THC-9-OG/5FU plus THC-9-OG to assess the extent of DDR activation and the WB results displayed substantial expression of XRCC1, RAD-51and MRE11 levels in alone 5FU treatment but 5-FU combined with THC-9-OG sharply depleted those DDR mediators consistently (Fig. 4A). On the other hand, pronounced DNA damage sensor γH2AX level was achieved following combination treatment compared to alone 5FU/THC-9-OG exposure. Further, to validate our immunoblotting results, following treatment with 5-FU, we performed comet tail assay showing alone 5-FU failed to initiate DNA damage within 36 h as evident from the insignificant comet tail formation. However, 750 nM 5FU combined with 3 µM THC-9-OG displayed significant formation of comet tail compared to vehicle, 750nM 5FU or 3µM THC-9-OG treatment (Fig. 4B). Additionally, to investigate the localization of DNA damage sensor γH2AX, immunocytochemistry analysis was carried out following exposure of HCT-116 cells to this combinatorial treatment and the results unleashed that γH2AX protein upon DNA damage tends to colocalize with ATM and MRN complex in its phosphorylated form, resulting in beads like structure in the nucleus when probed with γH2AX antibody in immunocytochemistry analysis (Fig. 4C).
The increased frequency of DNA damage upsurges chromosomal instability leading to cell death; thus, to study the effect of combinatorial treatment of 5FU and THC-9-OG on chromosomal stability, HCT-116 cells following treatment with indicated conditions were subjected to chromosomal preparation. The results demonstrated that combinatorial treatment (3 µM THC-9-OG plus 750 nM 5FU) elevated significant chromosomal instability resulting in various chromosomal aberration morphology viz breakage, chromosomal fusion, dicentric chromosomes compared to vehicle / 5FU / THC-9-OG treated samples (Fig. 4D & E). Together, these results convincingly described that tetrahydrocannabinol derivative in combination alleviated the DNA damaging propensity of 5FU (at sub toxic doses) and induced chromosomal instability.
Vimentin plays a key role in regulating the balance between DNA damage and repair
The activation of the EMT program during malignancy enhances the development of migratory behavior and affects plethora of integrated signaling networks related to cellular survival/anti-apoptosis 8. Emerging pieces of evidence suggest that EMT is linked with components of DDR and positively influence the DNA repair potential of the cancer cells, thus affecting the efficacy of DNA damaging drugs 30, 31, 32. Cytoskeleton protein Vimentin plays a pivotal role in maintaining cellular homeostasis by conducing the survival instincts of cells under threats including radiation or chemotherapeutics33, 34. Therefore, based on our above results, we were keen to examine any correlation between DNA damage sensors with Vimentin in the context of our combinatorial therapy. Our immunoblot results clearly revealed a reduction in Vimentin expression in THC-9-OG combined with 5FU treated lane compared to 5FU alone and surprisingly γH2AX sharply augmented by combinatorial treatment coupled with attenuation of repair protein Rad-51 at 36 and 48 h time points (Fig. 5A). Further, to understand that the increase in DNA damage by combinatorial treatment was sufficient to activate cell death, the expression of BCL-2 and BAX proteins were analyzed by immunoblotting. The immunoblot results demonstrated that colon cancer cells treated with 5FU + THC-9-OG showed a decreased level of BCL-2 and correspondingly the elevated expression of BAX at 36 and 48 h time points as compared to 5FU alone condition, thus indicating the onset of death signaling activation (Fig. 5A). Datasets were selected in STRING database and a potential relation was established between ATM, Zeb1 & Snail1. Vimentin and ATM were shown to be indirectly related via. Zeb1 and Snail1. Further, RAD51 and H2AX are mutually related to each other and exhibit direct relationship with ATM. These genes exhibited the relation that has been determined experimentally as well as through text-mining as shown in the Fig. 5B-D. cBioportal was accessed to study this relationship using TCGA atlas databases. mRNA co-expression was observed with the help of Illumina HiSeq tool and positive correlations were observed in diverse cancer models exploring TCGA databases. cBioportal helped to investigate the different cancer types with mutations and alterations in genes including ATM, Zeb1 & Snail1. These genes showed alterations and mutations in a wide range of cancer types. Spearman and Pearson distributions were used for statistical analysis along-with regression analysis to determine the correlation activity in diverse genes cluster. Potential mRNA expression correlation was determined along-with different gene alterations (as shown by different colors) between Zeb1 and ATM in a wide range of cancer types including Lung Squamous Cell Carcinoma and Colorectal Adenocarcinoma (TCGA PanCancer Atlas). Moreover, we analyzed and compared the mRNA expression between RAD51 and Snail-1. The TCGA pan cancer data mining results depicted a significant relationship between genes responsible for EMT (Snail-1) and DNA repair (RAD-51). (Supplementary Fig. 2).
To understand the inter-relationship between EMT and DDR, HCT-116 cells were transfected with GFP-Vimentin plasmid and subsequently treated with vehicle, 5FU and THC-9-OG for 48h. Following treatment, the cells were subjected to immunoblotting to study γH2AX, Rad-51, and Vimentin expression. Interestingly, the expression of γH2AX was significantly down modulated in lane treated with 5FU in combination with THC-9-OG in the presence of GFP-Vimentin compared to lane devoid of Vimentin overexpression. Correspondingly, the Rad-51, MRE-11and XRCC1 levels also amplified significantly in GFP-Vimentin overexpressed plus 5FU treatment compared to the 5FU + THC-9-OG treated lane (Fig. 5E). Hence, these results depicted Vimentin's importance in maintaining the critical balance between DNA damage and its repairing mechanism. In addition, Vimentin's abrogation with the THC-9-OG molecule shifted the balance towards extensive DNA damage at later time points (36–48 h). Similarly, the immunocytochemistry data showed a reduction in Vimentin, MRE11 and XRCC1 expression in THC-9-OG combined with 5FU treated HCT-116 cells compared to 5FU alone (Fig. 5F & G). Since Vimentin was corroborating with the DNA damage repairing mediators as we could see in the above section, we were curious to examine the localization of p53 tumor suppressor as a consequence of EMT as well DDR activation in presence and absence of Vimentin. The results showed that cytosolic confinement of p53 along with Vimentin in presence of alone 5FU or THC-9-OG whereas combination of 5FU + THC-9-OG conferred nuclear translocation of p53. Strikingly, transient knock down of Vimentin with SiVimentin triggered p53 translocation to nucleus by 5FU treatment. (Fig. 5H). Cumulatively, these results implied that combinatorial application of 5FU + THC-9-OG consistently abrogated Vimentin levels along with DDR mediators; further, drug-induced Vimentin levels play a pivotal role in controlling ATM-mediated DDR response and Vimentin depletion by THC-9-OG and 5FU combination conferred p53 shuttling to the nucleus that may be responsible to activate cell death in corroborating with ATM kinase.
THC9 in combination with 5FU instigates ROS mediated detrimental autophagy to modulate p-ATM-Vimentin axis
Classes of fugitive, extremely reactive, oxygen carrying moieties-reactive oxygen species (ROS) are known to induce DNA damage and alter DNA damage response. ROS plays a crucial role in triggering cell death pathways 35. ROS stimulates autophagy to facilitate other homeostatic responses via recycling and degradation of intracellular macromolecules. Also, there are several reports suggesting the induction of autophagic cell death driven by ROS generation 36, 37.
Cannabinoids are known to induce ROS generation 38, 39 ; rationally, we sought to explore any ROS induction in presence of THC-9-OG and our DCFDA staining results demonstrated that THC-9-OG or 5FU alone failed to stimulate significant ROS production compared to combinatorial (THC-9-OG + 5FU) treatment which triggered substantial ROS generation compared to as good as positive control H2O2 (Fig. 6A). To validate our ROS Fluorescence data, we conducted immunoblot analysis employing combinatorial (THC-9-OG + 5FU) treatment in presence/absence of ROS inhibitor NAC. The results shown that THC-9-OG in combination with 5FU resulted in the robust increase in the DDR sensor γH2AX expression whereas DNA damage repairing mediator XRCC1 as well as anti-apoptotic protein BCL-2 expression were diminished in presence of THC-9-OG plus 5FU (Fig. 6B). Interestingly NAC treatment rescued these cells from DNA damage mediated apoptosis as witnessed by decrease in the expression of γH2AX and increase in the BCL-2 expression (Fig. 6B). Moreover, tetrahydrocannabinol possesses the ability to activate or inhibit CB1 receptor activity 40, 41; accordingly we performed immunoblotting analysis of HCT-116 cells in presence or absence of CB1 agonist (ACEA) or antagonist (AM281) in a co-treatment setup with 5FU. Interestingly, we observed increase in the expression of γH2AX and decrease in Vimentin expression in agonist plus 5FU treated cells mimicking THC-9-OG plus 5FU treatment suggesting the involvement of CB1 receptor activation in our combinatorial approaches (Fig. 6C).
Autophagy, a lysosomal catabolic process, eliminates damaged or defective cellular materials and is initiated in response to various stress stimuli like hypoxia, nutrient deprivation and oxidative stresses. Excess reactive oxygen species (ROS) often known to trigger cell death pathways whether it could be apoptosis/necroptosis or autophagy mediated. An intricate crosstalk between autophagy and ROS has been implicated by the researchers indicating their dual role in either suppressing malignant transformation or promoting disease etiology42, 43.
Since we have obtained extremely high ROS levels following our combinatorial approach, therefore, we sought to examine whether this combinatorial treatment could have any effects on triggering autophagy. Following exposure of colon carcinoma cells to THC-9-OG plus 5FU, we performed western blotting of significant markers of autophagy viz, LC3B and p62. The results shown steady turnover of LC3B-1 to LC3B-II and reduction in the levels of p62 in combinatorial treatment (Fig. 6D). Furthermore, to validate our immunoblotting results, we performed LC3B puncta assay by fluorescence imaging showing pronounced puncta formation in HCT-116 cells when co-treated with 5FU and THC-9-OG derivative (Fig. 6E). Interestingly, these punctas were absent in the combinatorial treatment (5FU and THC-9-OG) along with ROS inhibitor NAC or ATM inhibitor KU60019 suggesting 5FU and THC-9-OG combination triggered autophagic cell death via ROS generation and ATM activation. Plethora of recent research suggests the activation of ATM (ataxia-telangiectasia mutated) protein by ROS and its involvement in autophagy activation 44, 45. Of note in the previous results, there was remarkable ROS accumulation on combinatorial treatment of 5FU and THC-9-OG derivative in HCT-116 cells, as well as the induction of autophagy. These deliberations led us to hypothesize that the co-treatment of 5FU and THC-9-OG might be inducing detrimental autophagy by inducing ATM activation. Therefore, we performed immunoblotting experiments of p-ATM, LC3B and p62 in presence or absence of NAC and ATM inhibitor (KU 60019) following co-treatment of the HCT-116 cells with 5FU and THC-9-OG. Interestingly, we observed that in presence of NAC or KU 60019 co-treatment of 5FU plus THC-9-OG derivative was unable to induce the activation of p-ATM and autophagy as seen by decreased LC3B I-II conversion and p62 degradation compared to Lane-2 wherein the combination resulted pATM activation along with diminished expression of survival mediators Bcl-2 and Vimentin (Fig. 6F). Intriguingly, Vimentin expression was diminished in presence of 5FU plus THC-9-OG along with NAC compared to alone NAC treatment wherein Vimentin level was substantial (Fig. 6F, Lane 3 vs Lane 4) suggesting the role of excessive ROS in downmodulation of Vimentin. Further, to validate our immunoblot analysis, we performed immunofluorescence experiment by overexpressing RFP-Vimentin construct followed by treatment with our combination of 5FU and THC-9-OG. The results shown a decrease in ROS fluorescence in presence of 5FU and THC-9-OG in RFP-Vimentin well whereas there was sharp increase in Vimentin fluorescence observed in 5-FU treatment (Fig. 6G). Together, these results suggest that 5FU in combination with THC-9-OG stimulated ROS production, which in turn caused robust ATM activation along with complete abrogation of EMT mediator Vimentin leading to autophagic cell death.
THC-9-OG in combination with 5-Fluorouracil triggers cell death to abrogate survival/EMT
The DNA damage response encompasses a wide variety of processes that ranges from arresting the cell cycle, DNA repair to apoptosis depending on the extent of the damage. From the previous results, THC-9-OG, a potential anti-metastatic molecule in combination with 5FU, augmented the DNA damaging capacity of 5-Fluorouracil by abrogating the activation of vital DDR factor, Rad-51, which is intricately linked with EMT induction. Our cell viability data demonstrated steady attenuation in HCT-116 cells growth and proliferation in a wide range of time points (36 h to 60 h) in presence of 5FU / THC-9-OG / and combination (Fig. 7A). Further, to understand whether the consistent increment in DNA damage by combinatorial treatment was sufficient to activate cell death, the expression of Bcl-2 and BAX proteins were analyzed by immunoblotting at 36 h, 48 h and 60 h to correlate the expressions of survival/death markers at early and late stage. Since EMT might be interfering to confer survival responses, we did not find significant cell death with our combinatorial treatment at 24 h (data not shown). The immunoblot results demonstrated that 5FU + THC-9-OG treatment showed a decreased level of Bcl-2 and corresponding increase in the of BAX expression at 36 and 48 and 60 h whereas the reverse trend of Bcl-2 and BAX expression was noticed at 36 and 60 h when treated with 5FU alone indicating induction of cell death activation by combinatorial treatment compared to stand alone drug 5FU (Fig. 7B & D). Furthermore, in the same experimental set up, HCT-116 cells treated with 5FU in combination with THC-9-OG for indicated conditions were subjected to immunoblotting to check the expression of cleaved Caspase-3, and Rad-51 protein and intriguingly the results showed a significant expression of cleaved Caspase-3 at 48 and 60 h time points in lanes treated with 5FU in combination with THC-9-OG as compared to alone 5FU / THC-9-OG treatment wherein the expression of cleaved Caspase-3 was depleted (Fig. 7B & C). Apart from cleaved Caspase-3, the elevated expression of Rad-51 was indicative of DNA damage repair was amplified at 36 and 60 h in presence of 5-FU whereas Rad-51 got diminished in combinatorial treatment at the same time point (Fig. 7B). These results demonstrate THC-9-OG ameliorates DNA damage propensity of 5FU to activate cell death by interfering with the DNA repair mechanism of colon cancer cells. Further, we performed flow cytometry analysis of HCT-116 cells following staining with Annexin-FITC and propidium iodide and post treatment with vehicle (DMSO) or 5FU plus THC-9-OG. Our results depicted huge induction of apoptosis in combinatorial treatment of 5FU and THC-9-OG compared to vehicle treated cells (Supplementary Fig. 3).
The combinatorial treatment efficacy was further studied in APC knockout colorectal carcinoma model wherein the crypt progenitor morphology was observed. The APC floxed mice were treated with vehicle, THC-9-OG (30mg/kg), 5FU (1.6 mg/kg), and THC-9-OG (30mg/kg) + 5FU (1.6 mg/kg) for 48h. Upon termination of the treatment period, the animals were sacrificed, and the entire gut was dissected out and further processed for H & E staining. The slides depicted that the cohorts treated with 1.6 mg/kg 5FU alone displayed more prominent fused crypt and villi morphology (crypt progenitor phenotype) than the vehicle treated mice indicating the induction of the early EMT features (Fig. 7E). Interestingly, the combinatorial treatment significantly reversed this phenotype, resulting in restoring atypical crypt/villi histology compared to control cohorts manifesting EMT induction in vivo. Furthermore, immunoblotting of the intestinal tissues was performed to study the expression of γH2AX, Rad-51, Bcl-2, BAX, and Cleaved Caspase-3. The immunoblot results showed that the combinatorial treatment elevated the expression of γH2AX and correspondingly attenuated Rad-51 levels suggesting the potentiation of DNA damaging effect of 5FU by THC-9-OG molecule (Fig. 7F & G). Moreover, the increased expression of BAX and down modulation of pro-survival factor Bcl-2 signified apoptosis activation, further confirmed by Cleaved Caspase-3 levels (Fig. 7F & G). Altogether, these data demonstrated the promising role of THC-9-OG in alleviating the efficacy of 5FU in triggering cell death pathways by augmenting DNA damage and suppressing survival mediators in vitro and in vivo.