The potential of Cannabis Sativa L. aerial plant parts extracts to reverse drug resistance in selected resistant lung- and colon cancer cell lines
Mangoato, Innocensia M.
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A major problem related to the successful application of chemotherapy in human cancer is anti-cancer drug resistance. Verapamil is one of the first drugs known to circumvent multidrug resistance (MDR), but its clinical application is limited by lack of efficacy in clinical trials, enhanced toxicity to normal cells and inhibition of cytochrome P450 enzymes resulting in pharmacokinetic interactions with increased host toxicity, thereby leading to severe adverse effects. Thus, this study was designed to evaluate the potential reversal of doxorubicin resistance by Cannabis sativa L. extracts using selected MDR expressing lung- and colon cancer cells in an in vitro test model. Firstly, the pulverized plant material was sequentially extracted with four organic solvents, in order of increasing polarity, starting with hexane, dichloromethane (DCM), DCM: methanol (1:1; v/v) and methanol, respectively. A water extract was prepared to simulate traditional preparation of the plant. Crude extracts were further fractionated by means of solid phase extraction (SPE) using the following eluting concentrations: 100% H2O, 25% acetonitrile (ACN), 50% ACN, 75% ACN and 100% ACN. The SPE yielded five fractions from each of the extracts. Qualitative phytochemical analysis performed on the pulverized crude plant material indicated the presence of glycosides, saponins, terpenoids, tannins, phytosterols and no flavonoids. Chemical fingerprinting of the C. sativa L. crude extracts, SPE fractions and cannabis standards was determined by liquid chromatography tandem mass spectrometry (LC-MS). The DCM- and methanol extracts were subjected to ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS) analysis while the DCM: methanol crude extract, SPE fractions, and cannabis standards (CBD and THC) were analysed using high performance liquid chromatography tandem mass spectrometry (HPLC-MS). Compound separation was achieved with a gradient mobile phase of distilled H2O with 0.1% formic acid (A): ACN (B) at a flow rate of 0.4ml/min. The mass spectrometer with electrospray ionization was operated in both negative and positive mode for the DCM- and methanol extracts to avoid the destruction of chemically sensitive compounds, negative mode for the DCM: methanol extract and positive mode for the SPE fractions. UPLC-MS analysis showed that the negative mode detected more peaks compared to the positive mode. The major peaks in the DCM extract with retention times of 10.38- (327.1967m/z), 11.31- (359.2227m/z), and 12.76 minutes (353.1766m/z) were also observed in the methanol extract, with only slight variation in the retention times at 10.37- (327.2158m/z), 13.68 (359.2227m/z), and 14.67 minutes (353.1758m/z). In the positive mode, only one peak in the DCM extract, with retention time of 12.96 minutes (282.2805m/z), was similarly observed in the methanol extract at a retention time of 14.06 minutes (282.2798m/z). Analysis of the DCM: methanol extract, SPE fractions, THC, and CBD revealed the presence of different compounds with different molecular weights. Some of the major peaks observed in both the DCM- and methanol extracts were also seen in the DCM: methanol extract. Chemical characterization of these peaks was not attempted but left for another project. Anticancer and cytotoxicity assays were conducted against a panel of human lung- and colon cancer cells, namely; HT-29, Caco-2, NCI-H146 [H146], HCT-15 MDR, LS513 MDR and H69AR MDR cells; and human normal colon (CCD-18Co) cells. According to the American National Cancer Institute (NCI) guidelines, plant extracts with IC50 values of less than 20μg/ml, between 20-100μg/ml and more than 100μg/ml are considered active, moderately active and inactive, respectively. Cytotoxicity results showed that DCM: methanol extract potently inhibited the growth of Caco-2, whilst moderately inhibiting the HCT-15, LS513 and NCI-H146 [H146] cells growth. The methanol extract showed moderate growth inhibition of LS513 and NCI-H146 [H146] cells, and potently inhibited the Caco-2 cells. The hexane extract showed good growth inhibition of Caco-2 cells; and moderately inhibited LS513, NCI-H146 [H146] and H69AR cells. Similarly, the DCM and H2O extracts showed good growth inhibition of Caco-2 and HT-29 cells, whilst moderately inhibiting the growth of HCT-15, LS513, NCI-H146 [H146], and H69AR cells growth. All the extracts appeared to be more cytotoxic towards all the lung- and colon cancerous cell lines than the normal colon cells as indicated by their selectivity indices. The resistant reversal effect of doxorubicin by C. sativa L. extracts was determined on Caco-2, HCT-15, LS513 and H69AR cells through combination of the extracts with doxorubicin. C. sativa L. extracts showed MDR reversal activities in HCT-15, LS513 and H69AR cells characterized by decreased IC50 values of the extracts. In Caco-2 cells, the hexane-, DCM-, DCM: methanol- methanol- and H2O extracts showed an increase in their IC50 values from 0.64-, 0.65-, 0.67-, 0.02- and 0.55μg/ml to 2.0-, 1.92-, 5.67-, 8.72- and 1.56μg/ml, respectively, and were 0.32-, 0.34-, 0.12-, 0.002- and 0.35-fold more sensitive to doxorubicin compared to verapamil with a 4.80-fold reversal factor. In contrast, the same extracts showed a reduction in their IC50 values from 180.5-, 140.4-, 47.08-, 140- and 25.6μg/ml to 39.33-, 40.13-, 1.45-, 1.89-and 12.3μg/ml and increased doxorubicin sensitivity in HCT-15 cells by 4.59-, 3.50-, 32.97-, 74.07- and 2.08-fold, respectively, compared to verapamil, which showed a 1.41-fold reversal factor. These extracts showed 2.2-, 300.7-, 9.1-, 4.3- and 11-fold more sensitivity to doxorubicin than verapamil with a 0.05-fold reversal factor in LS513 cells. These extracts were 0.32-, 0.34-, 0.12-, 0.002- and 0.35-fold sensitive to doxorubicin compared to verapamil with a 4.80-fold reversal factor. The same extracts also increased doxorubicin sensitivity in H69AR cells by 8.60-, 7.09-, 11.34-, 20.51- and 11.42-fold compared to verapamil that showed 0.87-fold reversal factor. The combination index (CI) analysis demonstrated that both the control and extracts yielded a normal to very strong synergistic interaction (CI<1) in Caco-2 cells, normal to strong synergistic interaction (CI <1) in HCT-15 cells, moderate to strong synergistic interaction (CI <1) in LS513 cells and nearly additive (CI=1) to antagonistic interaction (CI >1) in H69AR cells. Based on this evidence, the extracts were successful in increasing the sensitivity of HCT-15, LS513 and H69AR cells to doxorubicin in vitro. Future research is warranted to purify the most active extract and study the biological mechanisms involved in reversing doxorubicin resistance both in vitro and in vivo