Cannabidiol and Oxygen-Ozone Combination Induce Cytotoxicity in Human Pancreatic Ductal Adenocarcinoma Cell Lines

Simple Summary Pancreatic cancer (PC) is related to lifestyle risks, chronic inflammation, and germline mutations. Surgical resection and adjuvant chemotherapy are the main therapeutic strategies but are less effective in patients with high-grade tumors. Oxygen-ozone (O2/O3) therapy is an emerging alternative tool for the treatment of several clinical disorders. The advantages of using cannabinoids have been evaluated in several human cancers. Regarding PC, activation of cannabinoid receptors was found to induce PC cell apoptosis without affecting the normal pancreas cells. Herein, we evaluate the anticancer effect of cannabidiol (CBD) and O2/O3, alone or in combination, on two human pancreatic ductal adenocarcinoma (PDAC) cell lines, PANC-1 and MiaPaCa-2, examining expression profiles of 92 pancreatic adenocarcinoma associated genes, cytotoxicity, migration properties, and cell death. Finally, we assess the combination effects with gemcitabine and paclitaxel. Summarizing, for the first time the antitumoral effect of combined therapy with CBD and oxygen-ozone therapy in PDAC is evidenced. Abstract Pancreatic cancer (PC) is related to lifestyle risks, chronic inflammation, and germline mutations in BRCA1/2, ATM, MLH1, TP53, or CDKN2A. Surgical resection and adjuvant chemotherapy are the main therapeutic strategies but are less effective in patients with high-grade tumors. Oxygen-ozone (O2/O3) therapy is an emerging alternative tool for the treatment of several clinical disorders. O2/O3 therapy has been found to ameliorate mechanisms promoting chronic pain and inflammation, including hypoxia, inflammatory mediators, and infection. The advantages of using cannabinoids have been evaluated in vitro and in vivo models of several human cancers. Regarding PDAC, activation of cannabinoid receptors was found to induce pancreatic cancer cell apoptosis without affecting the normal pancreas cells. In a murine model of PDAC, a combination of cannabidiol (CBD) and gemcitabine increased survival length by nearly three times. Herein, we evaluate the anticancer effect of CBD and O2/O3, alone or in combination, on two human PDAC cell lines, PANC-1 and MiaPaCa-2, examining expression profiles of 92 pancreatic adenocarcinoma associated genes, cytotoxicity, migration properties, and cell death. Finally, we assess the combination effects with gemcitabine and paclitaxel. Summarizing, for the first time the antitumoral effect of combined therapy with CBD and oxygen-ozone therapy in PDAC is evidenced.


CBD Induces Apoptotic Cell Death in PDAC Cancer Cell Lines
To assess cell death, FITC-conjugated Annexin V and Propidium Iodide (PI) staining and cytofluorimetric analysis were used. After 48 h of daily treatment with CBD (12.5-25 μM), it was observed that CBD induces an increased percentage of cells undergoing apoptosis compared to control, in both cell lines. PANC-1 and MiaPaCa-2 showed a significant increase in apoptotic cell death with CBD 25 μM compared to 12.5 μM (Figure 2).

CBD Induces Apoptotic Cell Death in PDAC Cancer Cell Lines
To assess cell death, FITC-conjugated Annexin V and Propidium Iodide (PI) staining and cytofluorimetric analysis were used. After 48 h of daily treatment with CBD (12.5-25 µM), it was observed that CBD induces an increased percentage of cells undergoing apoptosis compared to control, in both cell lines. PANC-1 and MiaPaCa-2 showed a significant increase in apoptotic cell death with CBD 25 µM compared to 12.5 µM (Figure 2).

CBD Induces Apoptotic Cell Death in PDAC Cancer Cell Lines
To assess cell death, FITC-conjugated Annexin V and Propidium Iodide (PI) staining and cytofluorimetric analysis were used. After 48 h of daily treatment with CBD (12.5-25 μM), it was observed that CBD induces an increased percentage of cells undergoing apoptosis compared to control, in both cell lines. PANC-1 and MiaPaCa-2 showed a significant increase in apoptotic cell death with CBD 25 μM compared to 12.5 μM (Figure 2).  To confirm apoptosis, Caspase 3 (Casp3) activation was evaluated, by Western Blot analysis. Cells were treated with CBD 25 µM for 48 h in daily administration and the results confirm an increase in activated Casp3 in both cell lines, especially in MiaPaCa-2 cells ( Figure 3A). Moreover, by Comet assay analysis, we confirmed that the CBD 25 µM after 48 h of treatment induced DNA damage ( Figure 3B). To confirm apoptosis, Caspase 3 (Casp3) activation was evaluated, by Western Blot analysis. Cells were treated with CBD 25 μM for 48 h in daily administration and the results confirm an increase in activated Casp3 in both cell lines, especially in MiaPaCa-2 cells ( Figure 3A). Moreover, by Comet assay analysis, we confirmed that the CBD 25 μM after 48 h of treatment induced DNA damage ( Figure 3B).  Pro-Casp3 densitometric values were normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as loading control, Casp-3 densitometric values were normalized to Pro-casp3. Blots are representative of one of three separate experiments, * p < 0.05, ** p < 0.01, *** p < 0.001 treated vs. untreated cells. The whole western blot image can be found in Figure S11. (B) Cell damage and DNA fragmentation were determined on PANC-1 and MiaPaCa-2 cells untreated (Vehicle Vhc) and treated with CBD for 48 h by Comet assay (alkaline electrophoresis conditions 20 V for 10 min, image acquisition 10×).

CBD Reduces Cell Migration of PDAC Cell Lines
To examine the role of CBD in regulating migration of PANC-1 and MiaPaCa-2 cells, the wound-healing assay was performed. The results showed that CBD 12.5 µM, does not induce a significant effect in cell migration after 24 h of treatment, while at 48 h, a reduction of cell migration is observed in both cell lines ( Figure 4). These data suggested that CBD influences PDAC cell line migration.

CBD Reduces Cell Migration of PDAC Cell Lines
To examine the role of CBD in regulating migration of PANC-1 and MiaPaCa-2 cells, the woundhealing assay was performed. The results showed that CBD 12.5 μM, does not induce a significant effect in cell migration after 24 h of treatment, while at 48 h, a reduction of cell migration is observed in both cell lines ( Figure 4). These data suggested that CBD influences PDAC cell line migration.

CBD Increases Chemosensitivity in PDAC Cell Lines
In order to evaluate a synergistic effect between CBD and the most common chemotherapeutic drugs used in PDAC treatment, GEM (up to 800 μM) and PTX (up to 28 μM) were tested in both cell lines. The results evidenced that MiaPaCa-2 cells are more sensitive to GEM and PTX than PANC-1 and that PTX shows a higher cytotoxic effect than GEM, in both cell lines (PANC-1 GEM IC50: 143.8 ± 2.4 μM; PTX 33.57 ± 1.2 nM, MiaPaCa-2 GEM IC50: 63.6 ± 3.5 μM; PTX 21.18 ± 1.1 nM) ( Figure 5), at 72 h post-treatments.
Subsequently, PDAC cell lines were exposed to CBD at 6.25, 12.5, and 25 μM in combination with three doses of each chemotherapeutic drug (GEM 100, 50, and 25 μM, and PTX 7, 3.5, and 1.75 μM) for 72 h. Chemotherapeutic drugs were administered once, while CBD was administered daily. The results showed that CBD 6.25 μM, in combination with all tested doses of GEM and PTX, did not increased cytotoxic effects compared with chemotherapeutic drugs alone, while CBD 12.5 and 25 μM was able to increase the cytotoxic effect induced by both chemotherapeutic drugs alone ( Figure 6). Indeed, synergistic effects were obtained with PTX 7 μM especially in MiaPaCa-2 cells while, in PANC-1, this combination resulted in an additive effect. Combination with GEM 100 μM induced an additive and synergistic effect with, respectively, CBD 12.5 and 25 μM, in MiaPaCa-2 but not in the PANC-1 cell line ( Figure S3). The results evidenced that CBD, at the appropriate doses, should be useful to enhance the chemotherapeutic drugs effects.

CBD Increases Chemosensitivity in PDAC Cell Lines
In order to evaluate a synergistic effect between CBD and the most common chemotherapeutic drugs used in PDAC treatment, GEM (up to 800 µM) and PTX (up to 28 µM) were tested in both cell lines. The results evidenced that MiaPaCa-2 cells are more sensitive to GEM and PTX than PANC-1 and that PTX shows a higher cytotoxic effect than GEM, in both cell lines (PANC-1 GEM IC 50     Subsequently, PDAC cell lines were exposed to CBD at 6.25, 12.5, and 25 µM in combination with three doses of each chemotherapeutic drug (GEM 100, 50, and 25 µM, and PTX 7, 3.5, and 1.75 µM) for 72 h. Chemotherapeutic drugs were administered once, while CBD was administered daily. The results showed that CBD 6.25 µM, in combination with all tested doses of GEM and PTX, did not increased cytotoxic effects compared with chemotherapeutic drugs alone, while CBD 12.5 and 25 µM was able to increase the cytotoxic effect induced by both chemotherapeutic drugs alone ( Figure 6). Indeed, synergistic effects were obtained with PTX 7 µM especially in MiaPaCa-2 cells while, in PANC-1, this combination resulted in an additive effect. Combination with GEM 100 µM induced an additive and synergistic effect with, respectively, CBD 12.5 and 25 µM, in MiaPaCa-2 but not in the PANC-1 cell line ( Figure S3). The results evidenced that CBD, at the appropriate doses, should be useful to enhance the chemotherapeutic drugs effects.

O2/O3 Treatment Improves CBD Cell Cytotoxicity in PDAC Cells
The effect of O2/O3 on PDAC cell lines was evaluated by using a hypoxia chamber. PANC-1 and MiaPaCa-2 cell lines were daily treated with O2/O3 and cell viability, with respect to cells cultured in normoxia, was evaluated up to 72 h, by MTT. Additionally, the effect of CBD administration in O2/O3treated cell lines was evaluated. The results showed that the addition of O2/O3 strongly reduces cell viability, as observed starting from 24 h post-treatment, and the effect of O2/O3 was increased by CBD in a dose and time dependent manner, in both cell lines ( Figure 7). Furthermore, H6c7 and NHF were daily treated with CBD and O2/O3, as performed with PANC-1 and MiaPaCa-2 cell lines, and cell viability was evaluated at 72 h post-treatment. The results evidenced that O2/O3 did not influence H6c7 and NHF cell viability and did not increase the CBDeffects ( Figure S4). Summarizing, H6c7 and NHF cell lines resulted less sensitive to CBD and O2/O3 both administered alone and in combination, as described in Table 1. Table 1. IC50 values and percentages of viability in PANC-1, MiaPaCa-2, H6c7 and NHF cell lines with CBD and O2/O3, alone or in combination, expressed as mean ± SD of three separate experiments.  Furthermore, H6c7 and NHF were daily treated with CBD and O 2 /O 3 , as performed with PANC-1 and MiaPaCa-2 cell lines, and cell viability was evaluated at 72 h post-treatment. The results evidenced that O 2 /O 3 did not influence H6c7 and NHF cell viability and did not increase the CBD-effects ( Figure S4). Summarizing, H6c7 and NHF cell lines resulted less sensitive to CBD and O 2 /O 3 both administered alone and in combination, as described in Table 1. Moreover, cell death was examined at 24 h post-treatment, by Annexin V staining and PI incorporation assay and results showed that O 2 /O 3 is able to induce necrotic cell death in both cell lines, and that O 2 /O 3 -CBD treatment increases the percentage of PI positive cells ( Figure 8).   . Addition of O2/O3 improves cytotoxic effect of CBD and chemotherapeutic drugs. Cell viability was determined in PDAC cell lines by MTT assay, and the percentage of cell viability was calculated compared with O2/O3 alone. Vhc showed in the graph is O2/O3 alone. O2/O3 conditioned cells were treated for 24 and 48 h with CBD, alone and in combination with different doses of GEM and PTX. Data shown are expressed as mean ± SE of three separate experiments. * p < 0.05 vs. Vhc # p < 0.05 vs. CBD alone, ç p < 0.05 vs. chemotherapeutic drug alone.

Gene Modulation by CBD and O2/O3 Treatments in PDAC Cell Lines
To elucidate the molecular events induced by CBD and O2/O3 treatment, 92 genes involved in PDAC progression and aggressiveness were evaluated by Taqman Array. Both cell lines were treated with CBD, O2/O3, and with CBD plus O2/O3, and the molecular pathways of PDAC associated gene were evaluated. As shown, different pathways involved in PDAC carcinogenesis were modulated by CBD, O2/O3, and by their combination, suggesting that both treatments influence common but also specific pathways. Regarding the effect in regulating genes involved in cell cycle progression, the major effects of CBD, after 24 h of treatment, was in down-regulation of Cyclin A2 (CCNA2) ( Figure  10A, Figure S5A). The effect of O2/O3 should be considered as very effective in regulating cell cycle since all the Cyclins (CCNs), Cyclin Dependent Kinases (CDKs) and E2Fs (E2F1, E2F3) analysed were down-regulated after 24 h of treatments, while the CDKN2A expression was up-regulated, compared to vehicle-treated cells ( Figure 10B, Figure S5B). Moreover, the co-treatments with O2/O3 and CBD, confirmed the effect of O2/O3 and, additionally, further up-regulated CDKN2A with respect to O2/O3treated cells ( Figure 10B, Figure S5B). To support the modulation of cell cycle gene pathways by CBD and O2/O3, PI staining and cell cycle analysis was performed. As reported, CBD and O2/O3 alone

Gene Modulation by CBD and O 2 /O 3 Treatments in PDAC Cell Lines
To elucidate the molecular events induced by CBD and O 2 /O 3 treatment, 92 genes involved in PDAC progression and aggressiveness were evaluated by Taqman Array. Both cell lines were treated with CBD, O 2 /O 3 , and with CBD plus O 2 /O 3 , and the molecular pathways of PDAC associated gene were evaluated. As shown, different pathways involved in PDAC carcinogenesis were modulated by CBD, O 2 /O 3 , and by their combination, suggesting that both treatments influence common but also specific pathways. Regarding the effect in regulating genes involved in cell cycle progression, the major effects of CBD, after 24 h of treatment, was in down-regulation of Cyclin A2 (CCNA2) ( Figure 10A, Figure S5A). The effect of O 2 /O 3 should be considered as very effective in regulating cell cycle since all the Cyclins (CCNs), Cyclin Dependent Kinases (CDKs) and E2Fs (E2F1, E2F3) analysed were down-regulated after 24 h of treatments, while the CDKN2A expression was up-regulated, compared to vehicle-treated cells ( Figure 10B, Figure S5B). Moreover, the co-treatments with O 2 /O 3 and CBD, confirmed the effect of O 2 /O 3 and, additionally, further up-regulated CDKN2A with respect to O 2 /O 3 -treated cells ( Figure 10B, Figure S5B). To support the modulation of cell cycle gene pathways by CBD and O 2 /O 3 , PI staining and cell cycle analysis was performed. As reported, CBD and O 2 /O 3 alone slightly increased G2/M phase while the combination of both induced also a slight rise in Sub-G1 phase, in both cell lines ( Figure S6).
Cancers 2020, 12, x 11 of 23 slightly increased G2/M phase while the combination of both induced also a slight rise in Sub-G1 phase, in both cell lines ( Figure S6). Summarising, these data suggest that the co-treatment with O2/O3 and CBD was efficient in reducing cell cycle progression in PDAC cell lines, regulating several markers associated with cancer cell proliferation. Moreover, we found that CBD significantly reduced ETS Like-1 protein Elk-1 (ELK1), Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2), Mitogen-activated Protein Kinase Kinase 1 (MAP2K1), and RAF-1, while no effects were observed in Ras-pathways inhibitors, compared to vehicle treated cells ( Figure 11, Figure S7). Comparing O2/O3 treatments with vehicle-treated cells, we found that all the Ras-associated pathways genes were down-regulated, while no effect was observed for Rac Family Small GTPase 2 (RAC2) (Figure 11, Figure S7). The combination with CBD induced a total inhibition of BRAF gene expression and strongly increased MAP2K1, MAP2K2, and ERBB2 gene levels compared with O2/O3 alone. Furthermore, no significant additional effects on the others Rasassociated genes were observed ( Figure 11, Figure S7).
Thanks to Taqman Array we also detected that CBD significantly increases the TP53 gene, which is associated with DNA repair (Figure 12, Figure S8). O2/O3 treatment was effective in increasing BRCA2 and TP53 gene expression with respect to vehicle-treated cells (Figure 12, Figure S8). However, the combination with CBD reduced BRCA2 and TP53 gene expression, restoring the expression of untreated cells (Figure 12, Figure S8). Summarising, these data suggest that the co-treatment with O 2 /O 3 and CBD was efficient in reducing cell cycle progression in PDAC cell lines, regulating several markers associated with cancer cell proliferation.
Moreover, we found that CBD significantly reduced ETS Like-1 protein Elk-1 (ELK1), Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2), Mitogen-activated Protein Kinase Kinase 1 (MAP2K1), and RAF-1, while no effects were observed in Ras-pathways inhibitors, compared to vehicle treated cells ( Figure 11, Figure S7). Comparing O 2 /O 3 treatments with vehicle-treated cells, we found that all the Ras-associated pathways genes were down-regulated, while no effect was observed for Rac Family Small GTPase 2 (RAC2) (Figure 11, Figure S7). The combination with CBD induced a total inhibition of BRAF gene expression and strongly increased MAP2K1, MAP2K2, and ERBB2 gene levels compared with O 2 /O 3 alone. Furthermore, no significant additional effects on the others Ras-associated genes were observed ( Figure 11, Figure S7).  Regarding the Nf-kB pathway, CBD significantly increased RELB (Figure 13, Figure S7). O2/O3 treatment was effective in reducing NF-kB2, REL and Ras Homolog Family Member A (RHOA) gene expression with respect to vehicle-treated cells (Figure 13, Figure S9). The combination with CBD did not produce additional effects, except a further reduction in RHOA gene level. (Figure 13, Figure S9). Thanks to Taqman Array we also detected that CBD significantly increases the TP53 gene, which is associated with DNA repair (Figure 12, Figure S8). O 2 /O 3 treatment was effective in increasing BRCA2 and TP53 gene expression with respect to vehicle-treated cells (Figure 12, Figure S8). However, the combination with CBD reduced BRCA2 and TP53 gene expression, restoring the expression of untreated cells (Figure 12, Figure S8).  Regarding the Nf-kB pathway, CBD significantly increased RELB (Figure 13, Figure S7). O2/O3 treatment was effective in reducing NF-kB2, REL and Ras Homolog Family Member A (RHOA) gene expression with respect to vehicle-treated cells (Figure 13, Figure S9). The combination with CBD did not produce additional effects, except a further reduction in RHOA gene level. (Figure 13, Figure S9). expression with respect to vehicle-treated cells (Figure 13, Figure S9). The combination with CBD did not produce additional effects, except a further reduction in RHOA gene level. (Figure 13, Figure S9). Regarding Phosphatidylinositol 3-kinase/Protein kinase B (PI3K/AKT) pathway, CBD did not significantly modulate the expression of the main genes. Indeed, it induced a slight increase in PIK3CD and only PIK3CB, PIK3R1 and PIK3R2 were reduced. (Figure 14, Figure S10A). O2/O3 treatments reduced the expression of all analyzed genes compared with vehicle-treated cells ( Figure  14B, Figure S10B), and the combination with CBD maintained the effect of O2/O3 (Figure 14, Figure  S10).

Discussion
PDAC patients present for 80-90% non-resectable stage cancer or distal metastasis, so systemic chemotherapy is applied as first-line treatment [10,35]. This therapy includes GEM, PTX, and Regarding Phosphatidylinositol 3-kinase/Protein kinase B (PI3K/AKT) pathway, CBD did not significantly modulate the expression of the main genes. Indeed, it induced a slight increase in PIK3CD and only PIK3CB, PIK3R1 and PIK3R2 were reduced. (Figure 14, Figure S10A). O2/O3 treatments reduced the expression of all analyzed genes compared with vehicle-treated cells ( Figure  14B, Figure S10B), and the combination with CBD maintained the effect of O2/O3 (Figure 14, Figure  S10).

Discussion
PDAC patients present for 80-90% non-resectable stage cancer or distal metastasis, so systemic chemotherapy is applied as first-line treatment [10,35]. This therapy includes GEM, PTX, and

Discussion
PDAC patients present for 80-90% non-resectable stage cancer or distal metastasis, so systemic chemotherapy is applied as first-line treatment [10,35]. This therapy includes GEM, PTX, and nucleoside analogues [36] in monotherapy or in combination with radiotherapy or by a poly-chemotherapeutic regiment [37]. However, so far treatment efficacy in PDAC is considered limited. The interest associated with cannabinoids administration is related to their palliative effects useful for the treatment of cancer pain, but also for their activity as anticancer compounds able to induce inhibition of cancer cell growth and increasing cancer cell death. CBD anticancer effects, recently reviewed, were investigated in in vitro and in vivo models of glioma, leukaemia, breast, lung, thyroid, colon carcinoma, myeloma, and melanoma [25]. CBD activities are mediated through the binding to different receptors (CB1, CB2, and Gpr55 receptors), and some members of TRP channels family (as TRPV1-2-3-4, TRPM8 and TRPA1), but also by a receptor-independent mechanism [38,39]. We firstly profiled CBD-ligand receptors expression, evidencing low levels of CB1 in both cell lines, while CB2 receptor was more expressed in PANC-1 than in MiaPaCa-2. Previously, CB1 and CB2 expression was evaluated in four human pancreatic cancer cell lines and biopsies compared to normal pancreatic biopsies. Data showed different CB1 and CB2 expression levels in pancreatic cancer cell lines, compared to normal pancreatic tissues [40]. Moreover, according to our results, mRNA levels of CB1 and CB2 showed equal quantities of CB1 receptor in PANC-1 and MiaPaCa-2 cell lines and confirmed higher levels of CB2 receptor in PANC-1 respect to MiaPaCa-2 [41].
Up to now, the role of CBD in inducing anticancer effect in pancreatic cancer has not been well characterized. However, it was found that ∆9-tetrahydrocannabinol (THC) decreased PANC-1 and MiaPaCa-2 cell viability and induced apoptotic cell death both in vitro and in vivo [40]. Moreover, CBD through a GPR55 receptor antagonism was evidenced to reduce cell growth, cell cycle progression, and Mitogen-Activated Protein Kinase 1 (MAPK) signaling in different PDAC cell lines and the combination of CBD and GEM, was more effective compared with either treatment alone [33,34]. GEM and PTX are chemotherapeutic drugs normally used for pancreatic cancer therapy. In our study the combined treatment with CBD/GEM and CBD/PTX was able to reduce cancer cells viability, and showed an increased effect underlined using different doses of CBD and chemotherapeutic drugs. Our study also showed CBD's ability to reduce PANC-1 and MiaPaCa-2 migration and similar evidence was found in other cancer cell lines [26,42].
Regarding O 2 /O 3 therapy, evidence has been provided of the anti-cancer effect of local administration of ozonated water treatment in vitro and in vivo, in a mouse model of rectal cancer. The local treatment induced damages only in the tumor tissues by inducing necrosis without affecting normal tissues [43]. Moreover, the inhibitory effect of O 2 /O 3 treatment in two human neuroblastoma cell lines was analyzed [24]. In this study, it was demonstrated that O 2 /O 3 treatment was able to reduce cell growth and to arrest cell cycle at G2 phase, by inhibiting the expression and localization of cyclin B1/cdk1 in neuroblastoma cell lines. Additionally, it was also evidenced that O 2 /O 3 improved the responsiveness to Cisplatin, Etoposide, and GEM [24]. These results support our evidence in pancreatic cell lines in which O 2 /O 3 administration strongly reduced PDAC cells viability, inducing necrotic cell death, and it was shown that O 2 /O 3 treatment is able to act synergistically with the common chemotherapeutic drugs used in pancreatic cancer management, PTX and GEM. Thus, supported by these results, we focused our attention on CBD and O 2 /O 3 combination, since no data were available about this potential co-administration. We found that CBD plus O 2 /O 3 enhanced the effect of both single treatments in reducing cell viability and increasing cell death, with effects observed just after 24 h of co-treatments, suggesting that the early effect was predominantly attributable to O 2 /O 3 . CBD was previously reported to have less cytotoxicity in non-tumoral cells [44]. The process that regulates the lower effects of O 2 /O 3 in non-tumoral cells is not well investigated. However, different research papers evidenced that O 2 /O 3 has a protective effect in normal cells, while it is toxic for cancer cells [45,46]. Different whole-exome sequencing studies on PDAC elucidated the major mutations and somatic copy number alterations including KRAS, TP53, SMAD4, CDKN2A, and damage repair genes such as BRCA1, BRCA2, giving rise to high cell proliferation and migration, reduction of cell death and genomic instability [47][48][49]. Since very little information relating to the molecular pathways involved in CBD and O 2 /O 3 effects are available, we investigated which genes could be modulated in PDAC cell lines, by analyzing 92 PDAC associated genes. Regarding cell cycle and proliferation pathways, we found that CBD reduced the expression of CCNA2. O 2 /O 3 strongly inhibited most of the genes that stimulate cell cycle-all of the CCNs, CDKs, and E2Fs (E2F1, E2F3)-while CDKN2A expression was up-regulated. In addition, the co-treatments with O 2 /O 3 and CBD further up-regulated CDKN2A with respect to O 2 /O 3 -treated cells. Similarly, CBD was found to inhibit Ras-associated genes such as ERBB2, RAF-1, ELK1, MAP2K1 and these effects were maintained in co-treatments with O 2 /O 3 for ELK1 and RAF-1, evidencing another potential mechanism to reduce PDAC gene profile. These data suggested that CBD and O 2 /O 3 should have an important role in dis-regulating the Ras pathways and its downstream CDKs signaling, and that could have potential therapeutic implications, since this pathway is correlated with various oncogenic signals, such as proliferation, chemoresistance, and migration in PDAC, as recently reviewed [50]. In addition, it should be taken into consideration that pre-clinical data revealed pharmacological inhibition of Ras pathways and MAPK signaling results in compensatory activation of PI3K/Akt signaling and vice versa, both of them necessary for PDAC progression [51,52] and that independent of Ras pathways, PI3K/Akt is often elevated in PDAC and correlated to tumor cell survival [53,54]. Herein, we also evidenced that CBD and O 2 /O 3 , as single treatment or in combination, are able to reduce the expression of different genes of the PI3K pathway. PDAC, as above described, is also characterized by the acquisition of an anti-apoptotic phenotype, partially by inhibition of p53 and over-expression of anti-apoptotic proteins, which correlate with short survival and overall survival, particularly in Bcl-2 positive cases [55,56], and mutation in genes related to DNA damage response, such as BRCA1/2 [54,57]. Herein, we showed that CBD alone increased p53 and reduced BRCA2 gene expression. Moreover, the combination with O 2 /O 3 restored the expression of untreated cells. In different cancer models, it was demonstrated that BRCA2 leads to accumulation of DNA breaks, and results in activation of p53, which promotes cell cycle arrest and activation of cell death [58,59]. Moreover, in cancer, BRCA2 inactivation leads to pro-inflammatory cytokines production, that is a determinant for cancer cell survival [60], and several studies have investigated the expression profile of various cytokines in patients with PDAC and Nuclear Factor kB (NF-κB) activation pathways that have also been shown to be involved in pancreatic cancer development [61][62][63]. Moreover, inhibiting NF-κB and its downstream targets, lead to the inhibition of proliferation, angiogenesis, and invasion as reported in the PDAC mouse model [64]. Herein, the effects of CBD and O 2 /O 3 on Nf-kB pathways were analyzed, and the results evidenced that O 2 /O 3 alone and in combination with CBD was able to reduce NF-kB-related genes supporting their role in reducing inflammation and potentially cellular migration.
Summarizing, our results showed that CBD and O 2 /O 3 were both able to induce significant changes in the expression profile of genes strongly involved in PDAC leading to the inhibition of cell viability, invasion, and increasing cell death. Moreover, CBD and O 2 /O 3 was found to increase the anti-tumoral effects of Gemcitabine and Paclitaxel, suggesting that these combinations could have significant potential as an effective therapy for pancreatic cancer that can enhance the effect of chemotherapy and overcome chemoresistance.

Cell Lines
Human pancreatic ductal adenocarcinoma (PANC-1 and MiaPaCa-2) cell lines were purchased by Sigma Aldrich (Milan, Italy) and cultured in DMEM high glucose medium (EuroClone, Milan, Italy) supplemented with 10% of fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/mL penicillin, 100 mg streptomycin and 1 mM sodium pyruvate. Human Pancreatic Duct Epithelial H6c7 cell line was purchased by Kerafast (Boston, MA, USA) and cultured in Keratinocyte serum free medium, supplemented with epidermal growth factor and bovine pituitary extract. Normal Human Fibroblasts NHF cell line was purchased by IFOM (Rome, Italy) and cultured in DMEM supplemented with 10% of fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/mL penicillin, 100 mg streptomycin and 1 mM sodium pyruvate. Cell lines were maintained at 37 • C with 5% CO 2 and 95% of humidity.

RNA Isolation, Reverse Transcription and Quantitative Real-Time PCR and TaqMan Array
Briefly, total RNA from untreated or CBD and O 2 /O 3 treated cell lines was extracted using Rneasy Mini kit (Qiagen, Milan, Italy). One microgram of total RNA from each sample was subjected to reverse transcription in a total volume of 20 µL using the High-Capacity cDNA Archive Kit (Applied Biosystem, Foster City, PA, USA) according to the instructions. cDNAs were analyzed by qRT-PCR performed using an IQ5 Multicolor Real time PCR Detection system. Quantitative real-time polymerase chain reactions (qRT-PCR) were performed with QuantiTect Primer Assays (Qiagen) for Human Cannabinoid receptor 1 (CNR1, CB1), Human Cannabinoid receptor 2 (CNR2, CB2) and TRP (TRPV1, TRPV2, TRPV3, TRPV4, TRPM8, TRPA1), according to manufacturer's protocol. Measurement of GAPDH levels were used to normalize mRNA contents and target gene levels were calculated by 2 −∆∆Ct method. The TaqMan ® Array Human Pancreatic Adenocarcinoma 96-well Plate, containing 92 assays to pancreatic adenocarcinoma associated genes and 4 assays to candidate endogenous control genes, was purchased (Thermo Fisher, Grand Island, NY, USA) and used to evaluate the effects of the treatments in modulating PDAC-related genes. Measurement of two housekeeping genes (GAPDH; HPRT1; GUSB) on the samples was used to normalize mRNA content. The gene expression levels of treated cell lines were expressed as relative fold compared with untreated or vehicle-treated cells [65].

Reagents
Pharmaceutical grade Cannabidiol (CBD) crystals were purchased (Pharmacy S. Albano, Torino, Italy). CBD crystals were solubilized in ethanol 70% at 50 mM concentration. Paclitaxel (PTX; 6 mg/mL) and Gemcitabine (GEM; 50 µM) were purchased by Sigma Aldrich and solubilized in water. All the compounds were aliquoted and stored at −20 • C and each aliquot was used one time.

O 2 /O 3 Treatments
PANC-1 and MiaPaCa-2 cell lines were seeded on 96-well culture plates, or 12-well plates at a density of 3.0 × 10 4 cells/mL. The cells were pre-cultured in normoxia for 24 h. Subsequently, the culture plates were exposed twice to O 2 /O 3 treatments for 30 min in a Hypoxia Incubator Chamber (Stemcell Technology, Vancouver, BC, Canada), by injecting O 2 /O 3 until chamber saturation, using a Midi Ozon Active machine (Medica s.r.l., Bologna, Italy).
After treatment, the cell plates were replaced in the incubator at normoxia condition (37 • C with 5% CO 2 and 95% humidity) for 6 h and then, the O 2 /O 3 treatment was repeated as described. After that, the cells plates were maintained in normoxia for 24 up to 72 h, before performing the experiments.

MTT Assay
3 × 10 4 cells/mL were seeded in 96-well plates in a final volume of 100 µL/well. After one day of incubation, compounds or vehicles, alone or in combination, were added and six replicates were used for each treatment and all experiments were repeated three times. In some experiments, the treatment was daily added, after washing with fresh medium. After 24 or 72 h cell viability was investigated by adding 0.8 mg/mL of 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) (Sigma Aldrich) to the media. After 3 h the supernatant was removed, and the pellet of salt crystals was solubilized with 100 µL/well of DMSO. The absorbance of the sample against a background control was measured at 570 nm using an ELISA reader microliter plate (BioTek Instruments, Winooski, VT, USA).

Cell Cycle Analysis
Cells were seeded into 6-well plates (3 × 10 4 cells/mL) and treated with CBD (12.5 µM), in the presence and absence of O 2 /O 3 , for 12 h. Cells were fixed by adding ice-cold 70% ethanol for 1 h and then washed with staining buffer (PBS, 2% FBS and 0.01% NaN3). Next, cells were incubated with 100 µg/mL Ribonuclease A solution (Sigma Aldrich) for 30 min at 37 • C, stained with propidium iodide (PI) 20 µg/mL (Sigma Aldrich) at room temperature for 10 min and analysed on a FACScan flow cytometer using linear amplification and CellQuest software, version 3.0 (BD Biosciences, San Jose, CA, USA).

Cell Death Assay
Annexin V-FITC and PI staining followed by FACS analysis was used to evaluate cell death on PANC-1 and MiaPaCa-2 cancer cell lines. Cells at a density of 3 × 10 4 cells/mL were treated with different doses of CBD, in the presence and absence of O 2 /O 3 , for 24 up to 48 h. After treatment, cells were stained with 5 µL of Annexin V-FITC or with 20 µg/mL PI for 10 min at room temperature and then washed with binding buffer (10 mM N-(2-Hydroxyethyl)piperazine-N0-2-ethanesulfonic acid [HEPES]/sodium hydroxide, pH 7.4, 140 µM NaCl, 2.5 µM CaCl 2 ). The percentage of positive cells determined over 10,000 events was analysed with FACScan flow cytometer using the CellQuest software. All experiments were repeated three times.

Alkaline Comet Assay
Cell lines were plated in 6-well plates (3 × 10 4 cells/mL) one day before treatment exposure. Semi-confluent cultures were daily treated for up to 48 h with CBD 25 µM or vehicle. Cells treated with vehicle were included. All experiments were repeated three times. The comet assay was performed under alkaline conditions following the ABCAM protocol. Briefly, after exposure to treatments, the cells were resuspended in 1 × PBS and added to 75 µL of molten (37 • C) 0.5% low-melting-point agarose gel to achieve a cell concentration of 1 × 10 5 cells/mL. The agarose was pipetted onto the comet slides.
Slides were stored in the dark at 4 • C for 10 min before adding pre-chilled lysis buffer for 45 min at 4 • C in the dark. The slides were immersed in freshly prepared alkaline solution (0.25 M NaOH containing 0.1 µM EDTA, pH 12.6) for 30 min at the same conditions. Slides were then removed and washed twice with TBE buffer for 5 min. Gel electrophoresis was performed at 1 V cm −1 (length of the electrophoretic chamber) for 20 min (running amperage 3-5 mA with the distance between the two electrodes of 25 cm). The comet slides were washed with 70% ethanol for 5 min and air-dried for 1 h at room temperature. 100 µL of diluted SYBR Green solution was placed onto each dried agarose circle. The slides were then read with a fluorescence microscope (Leica Microsystems, Buccinasco, Italy).

Wound-Healing Assay
PANC-1 and MiaPaCa-2 cells were seeded onto a 24-well plate at a density of 4 × 10 4 /mL and 2.5 × 10 4 /mL, respectively. When cells were confluent, they were scratched using 10 µL sterile pipette tips, and medium was replaced with fresh medium containing low percentage of serum to minimize cell proliferation and prevent cell detachment and treated with CBD 12.5 µM. Images of wounded areas were taken at 0 h and 48 h. Images acquisition was carried out by a LeitzFluovert FU (Leica Microsystems) microscope. Remaining wound areas were determined using NIH Image J 1.44 software (Research Services Branch (RSB), National Institutes of Health (NIH), Bethesda, MD, USA) three separate distance measurements per well. Analyses were performed in triplicate.

Statistical Analysis
The data presented represent the mean with standard deviation (SD) of at least 3 independent experiments. Synergistic activity of the CBD and chemotherapeutic drugs combination was calculated by the Chou-Talalay method, which provides the theoretical basis for the combination index (CI)-isobologram equation. This method allows quantitative determination of drug interactions, where CI < 1, = 1, and > 1 indicates synergism, additive effect and antagonism, respectively. Based on these algorithms, computer software, CompuSyn 3.0.1 version (CompuSyn Software, ComboSyn, Inc., Paramus, NJ, USA, 2007) was used for automatically determining synergism and antagonism at all doses or effect levels [66]. The statistical significance was determined by Student's t-test and by One Way-Anova and Two Way-Anova with Bonferroni's post-test; * p < 0.05.

Conclusions
In conclusion, we found that CBD and O 2 /O 3 were both able to induce significant changes in the expression profile of genes strongly involved in PDAC, suggesting that further study on this combination could be addressed to better elucidate the role of CBD and O 2 /O 3 in the progression of PDAC.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/10/2774/s1, Figure S1: CBs and TRPs expression in PDAC cell lines. Figure S2: Human Pancreatic Duct Epithelial and Human Fibroblasts cell lines were more resistant to CBD, Figure S3: Isobologram plots and CI values for combination treatments of CBD, GEM and PTX in PANC-1 and MiaPaCa-2 cell lines, Figure S4: O 2 /O 3 did not increase CBD cytotoxicity in H6c7 and NHF cell lines, Figure S5: Modulation of cell cycle pathways in MiaPaCa-2 cell line. Figure S6: O 2 /O 3 , alone and in combination with CBD, reduced cell cycle progression in PANC-1 and MiaPaCa-2 cell lines. Figure S7: Modulation of Ras pathways in MiaPaCa-2 cell line, Figure S8: Modulation of DNA repair pathways in MiaPaCa-2 cell line, Figure S9: Modulation of NF-kB pathways in MiaPaCa-2 cell line, Figure S10: Modulation of PI3K/AKT pathway in MiaPaCa-2 cell line, Figure S11: Uncropped Western blot images from Figure 3 and Figure S1.

Conflicts of Interest:
The authors declare no conflict of interest.