Δ9–tetrahydrocannabinol (THC)-rich compositions from canna- bis have cytotoxic activity against ovarian cancer cells and act synergistically with niraparib in vitro

Ovarian cancer (OC) is the most lethal gynecologic malignancy. Cannabis sativa is being used to treat different medical conditions. We sought to examine the effectiveness of combinations of cannabis compounds against OC. Cytotoxic activity was determined by XTT assay on HTB75 and HTB161 cell lines. Apoptosis and cell cycle were determined by fluorescence-activated cell sorting (FACS). Gene expression was determined by quantitative PCR. The two most active fractions, F5 and F7, from a high Δ9–tetrahydrocannabinol (THC) cannabis strain extract and their standard mix (SM) showed cytotoxic activity against OC cells. The most effective phytocannabinoid combination was THC+cannabichromene (CBC)+cannabigerol (CBG). F5, F7 and SM affected cell cycle, led to cell apoptosis and to a marked reduction in cell migration. Moreover, these fractions act in synergy with niraparib, and were ~50 fold more cytotoxic to OC cells than to normal keratenocytes. Niraparib+F7 treatment was effective on OC patient's cells. F7 and the niraparin+fraction (F5 and F7) treatments reduced Mitogen-Activated Protein Kinase 4 (MAPK4) gene expression; this reduction may act in synergy with the niraparib inhibition of Poly (ADP-ribose) polymerase 1 (PARP1) activity. Combinations of cannabis compounds and niraparib should be examined for efficacy in pre-clinical studies and clinical trials.

pounds affected cell cycle and led to cell apoptosis. Moreover, these fractions act in synergy with niraparib in vitro; the synergetic mixture of niraparib+F5 or niraparib+F7 was ~50 fold more cytotoxic to OC cells than to normal keratinocytes.

Plant extraction
The dry inflorescence of the high Δ9-tetrahydrocannabinol (THC) C. sativa strain Dairy Queen (DQ) (IMC, Israel) was extracted as described previously [15]. Decarboxylation was carried out by heating the dry extract to 220 °C for 10 min. The heated extract was dissolved in methanol and filtered through a 0.45 µm syringe filter. Following evaporation, the weighted vials were re-suspended in methanol to the desired concentration.

Extract fractionation
A flash chromatography apparatus (Flash Pure, Buchi, C-810) equipped with a diode array detector was used to fractionate the decarboxylated crude extract. An Ecoflex C-18 80g, 50 µm spherical, max. pressure 180 psi column was used for separation, with 80-85% methanol in water as the mobile phase. The flow rate was 30 mL/min. The organic solvent (methanol) of each fraction was separately removed by using a rotary vacuum evaporator at 30 °C. The remaining aqueous phase containing the compound of interest was lyophilized to obtain a dried powder. Each dried fraction tube was weighed separately and reconstituted by methanol to produce the required concentrations, and stored at -20 °C.

Chemical analysis
High performance liquid chromatography (HPLC, 1260 Infinity II, Agilent) equipped with a Raptor ARC-18 for LC-UV column (150 mm × 4.6 mm ID, pore size 2.7 µm) was used for chemical analysis as previously described [16]. A gas chromatography-mass spectrometer (GC8860-MS5977B Agilent) equipped with 30 m, 0.25 mm ID, 5% crosslinked phenylmethyl siloxane capillary column (HP-5MS) with 0.25-μm film thickness, was used for chemical analysis as described in [16]. 10 μL of each sample fraction was transferred into GC vials with an insert, dried under a gentle stream of nitrogen and dissolved in 100 μL of hexane. Sample volume for injection was 1 μL. Helium was used as the carrier gas at a constant flow of 1.1 mL s -1 . An isothermal hold at 50 °C was maintained for 2 min, followed by a heating gradient of 6 °C min -1 to 300 °C, and the final temperature was held for 4 min. Peaks were assigned using spectral libraries (NIST 14.0 and 17.0) and compared with MS data obtained from the injection of standards purchased from LGC Standards.

Isolation of OC cells from patient MK
One cancerous deep femoral lymph node was removed from OC patient MK by a gynecological oncology surgeon. The node was placed in sterile saline solution, and a cold pack was activated for immediate overnight shipping to SEngine Precision Medicine (Seattle, WA, USA). The node was processed and grown into an organoid [17,18]. Genetic testing on MK's cells showed no germline or 2 somatic mutation, but showed mutant TP53 C135Y and STK11 splice site 735-1G>C. Immunohistochemistry of tumor indicates strongly positive for PAX8, WT1, negative for BRCA.

Cell Viability Assay
Cells were seeded into 96-well plates at density of 1 x 10 4 per well (in 50 µL/well) in complete medium and were incubated at 37 °C incubator overnight to allow attachment. The following day, cells were treated in triplicate with plant extracts, fractions, or cannabinoids standards in volume of 50 µL/well at different concentrations, as mentioned in each experiment. Solvents (methanol and/or DMSO) were used as a negative control and niraparib was used as a positive control in all the biological assays. In experiments where CB1 or CB2 inverse agonists, TRPV1 or TRPV2 antagonists, or TRPA1 blocker were used, they were added along with the treatment at concentration of 10 µM/mL. Treated cells were incubated for 48 h at 37 °C. Subsequently, XTT reagents (2,3,-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt) (20-300-1000, Biological Industries, Israel) were added to the cells for 2 h at 37 °C in a humidified 5% CO2-95% air atmosphere. Absorbance was recorded using a Synergy H1 hybrid reader photometer (BioTek) at 490 nm with a 650 nm reference wavelength. Cell viability was estimated using the equation: % = 100 × (( 490 − 650) ÷ ( 490 − 650) ). A490 and A650 are the absorbencies of the XTT colorimetric reaction. Absorbance of the media alone (blank) was substracted from the readings.
For dose response assay, GraphPad Prism version 6.1 (https://www.graphpad.com/scientific-software/prism/, GraphPad Software Inc., San Diego, USA) was employed to produce dose-response curves (data points were connected by non-linear regression lines of the sigmoidal dose-response relation) and determination of IC50 values. For assay of full confluence, cells were seeded in 96-well plates at density of 1 x 10 4 per well (in 100 µL/well) in complete medium and were incubated at 37 °C for 5 days to allow 100% confluence.

Apoptosis Assay
Apoptosis for HTB-75 cell line was assessed using the MEBCYTO Apoptosis Kit with Annexin V-FITC and PI (MBL, Enco, 4700). Staining was carried out according to manufacturer instructions. In brief, cells were seeded in a 6-well TC plates at a density of 5 × 10 5 cells in 2 mL medium per well, 24 h before treatment. The treatment period was 48 h, and cells were harvested after applying 250 μL trypsin for 5 min, adding a complete medium to neutralize the trypsin, and centrifuging for 5 min at 1600 rpm. Cell pellets were resuspended and washed twice with 1 mL of phosphate buffered saline (PBS). The cells in each sample were resuspended in 85 μL of Annexin binding buffer. Cells were stained with 10 μL of Annexin V-FITC solution and 5 μL of propidium iodide (PI) working solution followed by incubation in the dark at room temperature for 15 min. Next, 400 μL of Annexin V binding buffer was added to each tube and flow cytometry was performed using a Gallios flow cytometer (FACS). Cells were considered apoptotic if they were Annexin V+/PI-(early apoptosis) or Annexin V+/PI+ (late apoptosis). Live cells were defined as Annexin V-/PI-, and Annexin V-/PI+ as necrotic.

Cell cycle analysis
HTB-75 (CAOV-3) cells were seeded in 6-well TC plates at a concentration of 5 × 10 5 cells in 2 mL medium per well, 24 h before treatment. The treatment period was 48 h, and cells were harvested after applying 250 μL trypsin for 5 min, adding a complete medium to neutralize the trypsin, and centrifuging for 5 min at 1600 rpm. The cell pellet was washed once with 1 mL of PBS and fixed with 70% cold ethanol overnight at -20°C. The fixed cells were washed twice with 1 mL of PBS and stained with 250 μL of PI solution (40 μg/mL) containing RNase A (100 μg/mL) for 30 min in dark. 250 μL of PBS was added to each tube and the cells were analyzed using FACS.

Scratch-wound Assay
HTB75 cells were seeded into a 96-well plate at a density of 2 × 10 4 per well in 100 µL of complete medium. After 24 h, cells in each well were scratched perpendicularly across the center of the well with a 200 µL pipette tip to produce a cell-free area for investigating the ability of the cells to migrate and close the gap under different treatments. Immediately after scratching, 100 µL of treatment solution was added. Photos were taken at 0, 24, 30, and 48 h following scratching, and the gap area was measured using ImageJ (version 1.53a). The scratch area, indicated by cells migrated into the scratch was calculated as percent of scratch area at time x from time 0: ( h cell free area) × 100 (0 h cell free area)

Analysis of combined drug effects
Drug synergy was determined by the Bliss independence drug interaction model as described in [16], on HTB75 cells. Briefly, drug synergy was determined from XTT results using the Bliss independence drug interaction model, defined by the following equation: = + -( ), where (Exy) is the additive effect of the drugs x and y as predicted by their individual effects (Ex and Ey).

Quantitative real-time PCR
Cells were seeded in a 6-well plate at a concentration of 1.5 × 10 6 cells in 3 mL medium per well. After 24 h incubation, cells were treated with cannabis compounds and/or chemotherapy drugs for 6 h. Cells were harvested and RNA was extracted using TRI reagent (Merck). RNA was reverse-transcribed in a total volume of 20 μL (PB30.11-10, qPCRBIO) according to manufacturer's protocol. PCR was performed in triplicate using a qPCR Sy-Green Blue Mix (PB20.16-20, qPCRBIO) and StepOnePlus system (Applied Biosystems). The expression of each target gene was normalized to the expression of HPRT mRNA in the 2 -ΔΔCt method presenting the differences (∆) in threshold cycle (Ct) between the target gene and HPRT gene. ΔΔCt= ΔCt treatment-ΔCt control. Experiments were repeated three times. The primers included MAPK4 (Gene ID: 5596) (forward) 5'-TGTCAATGGTTTGGTGCTGT-3' and (reverse) 5'-TTTGACGATGTTGTCGTGGT -3'.

Statistical Analysis
Results are presented in graphs as mean + standard error (SE) of replicate analyses and are either representative of, or include at least two independent experiments. Means of replicates were subjected to the Tukey-Kramer test using the JMP statistical package (https://www.jmp.com/en_us/home.html, SAS Inc, NC, USA) and considered significant when P≤0.05.

Determining cannabis strain with cytotoxic activity against ovarian cell lines
Extracts of several C. sativa strains were examined for cytotoxic activity against ovarian cancer cell line HTB75. DQ (IMC, Israel), a high Δ9-tetrahydrocannabinol (THC) strain, was the most effective with the highest examined concentration (20 µg/mL) resulting in ~50% cell death (Figure 1a

Identification of active fractions of DQ cannabis strain
The most active extract, DQ, was fractionated as described in [15]. The activity of the fractions was tested on HTB75 cells. Four of the 11 fractions (F4, F5, F6 and F7) showed significant cytotoxic activity at the examined concentrations. F5 and F7 were the most active, at the highest examined concentration (20 µg/mL) resulting in ~80% cell death ( Figure  2a). F6 and F4 were less active, at the highest examined concentration (20 µg/mL) resulting in ~60% and ~40% cell death, respectively (Figure 2a). Treatments with F1-F3 and F8 showed no cytotoxic activity against HTB75 cells (Figure 2a). The IC50 of the most active fractions were determined to be 18.36 and 17.79 µg/mL for F5 and F7, respectively ( Figure  2b,c).

Determining activity of the phytocannabinoids standard mixes of F5 and F7
In order to confirm the active compositions of F5 and F7, standard mixes (SM) of phytocannabinoid standards were formulated as close as possible to the original fraction compositions. F5-SM had an IC50 of 14.67 µg/mL (Figure 3a), which was lower than that of F5 (18.36 µg/mL; Figure 2b). F7-SM showed IC50 value of 13.56 µg/mL (Figure 3b

Determining the most effective combination of phytocannabinoids
To determine the most effective combinations of the main phytocannabinoids [8] in F5 and F7, combinations of THC, CBG, CBC and CBN standards were examined at ratios found in F5 or F7, at fixed total concentration in comparison to THC (Figure 4). At concentrations of 13 and 15 µg/mL ( Figure 4a and Figure 4b, respectively), THC alone was less effective than some of the various combinations of THC, CBC, CBG and CBN at ratios found in F5 or F7 (Figure 4). The most effective combinations at both 13 and 15 µg/mL were those of THC+CBC and THC+CBC+CBG at ratios found in F7 (ratios of 7.5:2.5 and 7.4:2.5:0.1, respectively; Figure 4). For the F5 combination-ratio, THC+CBG was the most effective treatment, but was not significantly different from the other F5-based combinations ( Figure 4).

Determination of the involvement of CB1 and CB2 receptor inverse agonists, TRPA1 receptor blocker, and TRPV1 or TRPV2 receptor antagonists on cytotoxic activity
We determined the effect of adding CB1 or CB2 inverse agonists (IA), TRPV1 or TRPV2 antagonists (AN) or a TRPA1 blocker (B) on F5, F7, F5-SM and F7-SM activity. HTB75 cells were treated with F5, F7, F5-SM or F7-SM with or without IA, AN or B. In the presence of CB2 IA, the cytotoxic effect of F5, F7, F5-SM or F7-SM was significantly reduced (45.8, 62.7, 77.3, 57.1%, viable cells respectively, vs. 38.5, 19.3, 29.6, 26.3% viable cells without CB2 IA); Figure 6a-d). Also, TRPV2 AN co-treatment with F7 and F7-SM interfered to some extent with their activity (significantly with F7; Figure 6b,d). Cell viability percentage for treatments with F7 or F7-SM were 48.9 or 39.2% with TRPV2 AN vs 29.6 or 26.3% without, respectively (Figure 6b,d). Treatments with F5, F5-SM, F7 or F7-SM in the presence of TRPV1 AN, TRPA1 B or CB1 IA, did not significantly reduced the cytotoxicity of the treatments (Figure 6a-d). Treatment with TRPV1 AN even increase to some extent the cytotoxicity of F5-SM (Figure 6c). CB1 or CB2 IA, TRPV1 or TRPV2 AN or TRPA1 B did not affect HTB75 cell viability (Figure 6e).

Determining the effects of combining the most active cannabis fractions and chemotherapy drugs
Chemotherapy drugs were examined for combined activity with F5, F7, F5-SM and F7-SM. Considerable and significant synergy (on a scale of 0 [no synergy] to 1 [high synergy]) was obtained for combinations of niraparib with F5 (with peaks of 0.6-0.8 and significant synergy at 15 or 17.5 µg/mL F5 and 6-8 µg/mL niraparib; Figure 7a; Supplementary Table S3). Considerable and significant synergy was obtained also with combinations of niraparib and F7 (with peaks of 0.4-0.6, respectively, and significant synergy of 15 or 17.5 µg/mL F7 and 3-8 µg/mL niraparib; Figure 7a; Supplementary Table S3). Synergy with F5-SM or F7-SM on the other hand was restricted to 0.2-0.4 only (Figure 7a; Supplementary Table S3).
Gemcitabine showed only low level of synergy with the plant fractions (peaks of 0-0.2), and in some cases inhibited activity (e.g., for 12.5 µg/mL F5 or F7 and 5-100 ng/mL gemcitabine; Figure 7b; Supplementary Table S4) Supplementary Table S3 and Supplementary Table S4 are the different letters that signify different levels of the experimental (observed) values from all combinations of pairs according to the Tukey-Kramer honest significant difference test (n=3; HSD; P ≤ 0.05), for the synergistic delta values of F5, F7, F5-SM or F7-SM with niraparib or gemcitabine, respectively.

Determining the synergistic activity on normal cells
The cytotoxic activity of the synergistic mixture of niraparib+F5 and niraparib+F7 was assessed on normal cells using the keratinocyte (HaCaT) cell line. The synergistic ratio of mixture of niraparib:fraction (~0.53:1.00, derived from 8 µg/mL niraparib + 15 µg/mL F5 or F7; Figure 7a) was examined at several different concentrations; activity was examined in parallel on HaCaT and HTB75 cells at complete confluence ( Figure 8). Some minor cell proliferation was apparent in HaCaT but not in HTB75 cells under treatments with low concentrations (Figure 8). At the cytotoxic concentrations of the mixture of synergistic-ratio of niraparib+F5 or niraparib+F7 (i.e., 8.7 µg/mL niraparib + 16.3 µg/mL F5 or F7 and 10.4 µg/mL niraparib + 19.6 µg/mL F5 or F7) considerably higher cytotoxicity was apparent against the cancer cells than against normal cells. In the most effective combined treatments of niraparib+F5 or niraparib+F7 (10.4 µg/mL niraparib + 19.6 µg/mL F5 or F7) this activity was ~50 fold higher on HTB75 than on HaCaT cells (Figure 8).

Determining the synergistic activity on OC patient's cells
The cytotoxic activity of the synergistic mixture of niraparib+F5 and niraparib+F7 was assessed on OC patient's cells (MK), isolated from a cancerous deep femoral lymph node. These cells showed only low sensitivity to niraparib and no sensitivity to a combination of niraparib and F5 (Figure 9). However, a combined treatment of niraparib+F7 led to substantial cell death (Figure 9).

Determining the effect of synergistic and individual treatments of cannabis fractions and chemotherapy drugs on cell migration
The effect of the synergistic mixture of F5, F7, niraparib+F5 and niraparib+F7 on cell migration was examined at sub-lethal concentrations using a scratch-wound assay. Almost full closure of the scratch was obtained after 48 h in the vehicle control (10.1% of clear area; Figure 10). Niraparib inhibited wound closure, most apparent at 48 h (42.7% clear area; Figure 10). F5 treatment (30.4%) and F7 treatment (29.4%) inhibited scratch closure less than that of niraparib ( Figure 10). However, the combinatory treatments of niraparib + F5 or niraparib + F7 substantially inhibited cell migration, mainly at 48 h (50.9 and 54.7% clear area, respectively; Figure 10).

Determining MAPK4 expression in the various synergistic and individual treatments of cannabis fractions and chemotherapy drugs
MAPK4 gene expression was significantly reduced upon niraparib or F7 treatments in comparison to control ( Figure 11). F5 treatment did not reduce this gene expression ( Figure 11). In the synergistic treatments of niraparib+F5 or niraparib+F7 expression was reduced further, significantly in comparison to the individual treatments ( Figure 11).

Discussion
We have identified two compositions of phytocannabinoid mixtures from the extract of a high-THC cannabis strain with significant cytotoxic activity against OC cell lines, HTB75 and HTB161, both high-grade serous ovarian cancer [19]. To identify the active compounds, the crude extract was fractionated and active fractions were identified. Unlike in other studies [15,16], the active fractions exhibited only a minor advantage for cytotoxic activity over the crude extract.
The fractions contained both phytocannabinoids and terpenes. However, once phytocannabinoid standards alone were used, in the ratio found in each of the fractions (i.e., F5-SM, F7-SM), cytotoxic activity increased substantially. These results suggest that the active compounds for the cytotoxic activity against OC cell lines are the phytocannabinoids, and the terpenes or other unidentified compounds present in both the crude extract and fractions may interfere with this activity.
The most active fraction, F7, contains THC and CBC at approximately a 3:1 ratio, and CBG, CBDV and CBN in small amounts. F5 contains 91% of THC, and small amounts of CBG, THCV and CBN. Following, the most effective combinations of the main phytocannabinoids [6,8] for cytotoxic activity against OC cells were determined. Combinations of THC and CBC as in F7 were the most effective, with or without CBG. In F5 combinations, that of THC+CBG was highly active. These combinations were more effective than THC only.
Another fraction that contained THC, i.e., DQ-F6 [15], had cytotoxic activity against OC but to a lesser extent than F5 or F7. F6 contained more THC in relation to CBG in comparison to F7, and contained no CBC [15], which may explain its lower activity.
In multiple studies, THC and CBG have been suggested to possess anticancer activity for a variety of cancer types, in vitro, in vivo and for THC also in clinical trials [20]. CBC was shown to have anticancer activity on prostate carcinomas [21]. In addition, we previously found that a combination of THC and CBC was highly active against urothelial carcinoma cells [22]. Together, this study and others (e.g., [15,16,22]), highlight the im-portance of the appropriate combination of compounds for high specific anti-cancer activity in vitro; in the case of cytotoxicity to OC cells, a combination of THC and CBC with or without CBG.
Treatment with F5, F7, F5-SM or F7-SM led to an increase in apoptotic cell death in HTB75 cells. In multiple previous studies, phytocannabinoids were shown to induce apoptosis in cancer cells and to inhibit cancer cell proliferation [9,11]. Cell cycle arrest resulted in apoptosis in many cases [23]. In our study, cannabis treatments led to a significant increase in G2/M or S arrest, suggesting cell cycle arrest.
CB2 IA significantly blocked the cytotoxic activity of F5, F7 and their corresponding standard mixes. CB2 was overexpressed in malignant endometrial carcinoma cells but not in healthy cells [24].
The TRPV2 antagonist reduced the cytotoxic activity of F7 and F7-SM to some extent, suggesting that TRPV2 might be involved in the activity of these compounds on HTB75 cells. A study that examined protein expression in cancer tissues and adjacent normal tissues of OC patients revealed that TRPV2 is expressed in OC tissues at higher levels than in adjacent normal tissues. A significant increase in TRPV2 expressions in OC was also correlated with severity of tumor growth according to clinical stage [25]. Taken together, the higher level of TRPV2 and CB2 expression in malignant tissues may position TRPV2 and CB2 as target-receptors for cannabis-based therapy.
Neither the TRPA1 blocker, the TRPV1 antagonist nor the CB1 inverse agonist reduced activity, suggesting TRPA1, TRPV1 and CB1 are not involved in the cytotoxic activity of these compounds on HTB75 cells.
A plausible approach for treatment of OC patient is to combine the cannabis-based treatment with chemotherapy. Hence, we examined the effect of co-treatment of cannabis and chemotherapy drugs on HTB75 cell viability. Niraparib is a potent Poly (ADP-ribose) polymerase (PARP)-1 and PARP-2 inhibitor. PARPs are a family of nuclear proteins that allocate single-strand breaks in DNA, bind and activate recruitment of repair factors. These processes are important for cell proliferation. PARP inhibition by niraparib leads to cell cycle arrest and cell apoptosis [26,27]. Niraparib treatment led in our study to S phase arrest; indeed, PARP inhibition was previously shown to converts single strand breaks to double strand breaks during DNA replication in the S phase of the cell cycle [26], yet other studies niraparib was shown to induce cell cycle arrest mainly in the G2/M phase [27].
We found that niraparib acts synergistically with F5 and F7 at some of the examined concentrations, and is less synergistic with the standard mixes F5-SM or F7-SM. The fact that the plant fractions, containing both phytocannabinoids and terpenes, acted synergistically with the chemotherapy drugs, while the phytocannabinoids alone exhibited less, suggests that terpenes or other compounds present in the fractions facilitate this synergy. Gemcitabine did not show substantial synergistic activity with the cannabis-based treatments.
Moreover, the mixture of niraparib+F5 or niraparib+F7 at the synergistic ratio was ~50 fold more cytotoxic to HTB75 cells than to normal keratinocytes (HaCaT cells), once examined under complete confluence (to reduce non-cancerous cell proliferation in control treatment). The low cytotoxicity to normal cells may suggests that treatment with F5 or F7 in combination with niraparib could prove to be an effective and cancer-specific treatment. Supporting this suggestion, the combination of F7+niraparib showed activity on an OC patient's cells isolated from a cancerous deep femoral lymph node.
Niraparib or F7 treatments led to substantial reduction in MAPK4 expression. Members of the MAPK family are key regulator of cell proliferation, growth, and survival processes, and have implications in the response to cancer therapy [28]. MAPK4 is suggested to regulate DNA damage repair [29] and to promote tumor progression via noncanonical activation of AKT/mTOR signaling [30]. Specific to OC, MAPK4 upregulation was found to enhance cell proliferation and migration of cancer cells [31]. Importantly, it was shown that silencing MAPK4 expression in triple-negative breast cancer cells could enhance the sensitivity of these cells to PARP1 inhibitors and reduce the possibility of PARP1 inhibitor resistance [29]. Combined treatment including MAPK4 silencing and the PARP1 inhibitor olaparib inhibited cell proliferation and promoted apoptosis in these cells [29]. Also, in cervical cancer cells, MAPK4 knockdown and PARP1 inhibition exerted synergistic effects by activating AKT phosphorylation [32]. This suggests that the observed synergy between niraparib and the phytocannabinoids occurs because suppression of MAPK4 expression that enhances the PARP inhibitor cytotoxic activity on OC cells.
Reduction or abolishment of MAPK4 expression is associated with reduction in cell migration [29,31]. Overexpression of PARP1 promoted the wound-healing process in non-small cell lung cancer [33] and a combined treatment of the PARP1 inhibitor olaparib and silencing of MAPK4 expression further reduced cell migration in triple-negative breast cancer cells [29]. In agreement, we show here that treatment of OC cells with F7 that significantly inhibit MAPK4 expression, reduced cell migration, and that the co-treatment of F5 or F7 with niraparib reduced cell migration even more.
However, the fact that only niraparib+F7 treatment and not niraparibe+F5 was effective on MK cells suggests that the reduction of MAPK4 expression by itself is not sufficient to convey cell death of OC cells isolated from a cancerous deep femoral lymph node. Additionally, F5 treatment did not lead to reduction of MAPK4 expression but inhibited cell migration. Differences in mechanism of activity between F5 and F7 should be further examined.
To conclude, we identified cannabis compounds with substantial cytotoxic activity against OC cells in vitro, which involves cell cycle arrest and apoptosis. This activity was found to be considerably stronger on cancer cells than on normal cells. Indeed, CB2 and TRPV2, which might be involved with the compound's activity, are expressed more often in malignant tissue. Hence, cannabis-based therapies targeting cancer cells with reduced effect on the healthy surrounding tissues may be envisioned. Moreover, we identified synergistic activity between the cannabis extract fractions and niraparib. Since F5 and F7 treatments substantially reduced MAPK4 expression, this synergistic activity might be based on the known synergy between the repression of MAPK4 expression and the inhibition of PARP1 activity. Our results indicate that cannabis might be regarded as a complementary and effective anti-cancer treatment for OC. Given the favorable safety profile of phytocannabinoids compared to standard pharmacotherapy (e.g., [34]), we propose that clinical trials with cannabis-based products are needed desperately for OC patients. the manuscript. The final draft was completed by HK, and all authors read and approved the final manuscript.