LEY GENERAL DE AGUAS

Doxorubicin is clinically used for the treatment of a great variety of cancer disease (Gillle et al., 2002; Carter, 1975 and Khana et al., 1998). It has been one of the most extensively used agents in the chemotherapy regimens of cancer patients for the past 30 years (Weiss, 1992). In spite of the routine use of this drug its major adverse effect, the dose-dependent cardiotoxicity, cannot be prevented yet (Gillle et al., 2002). In the present study, doxorubicin was used as the positive control for the cytotoxicity assay. Thus, doxorubicin was screened for its cytotoxic activity on the selected human cancer cell lines, such as KB, CasKi, HCT 116, MCF7, A549 and non-cancer MRC-5 cell line.

Based on the result, it can be concluded that doxorubicin is not only cytotoxic against all the human cancer cell lines tested, but also the non-cancer human MRC-5 cell line (Table 4.16 and Figure 4.26). Doxorubicin possessed very strong cytotoxicity with IC50 values of 0.0125, 0.0060, 0.3600, 0.0755, 0.2200 and 0.5500 µg/ml against

167 0 10 20 30 40 50 60 70 80 90 100 0 0.2 0.4 0.6 0.8 1 Pe rc en ta ge o f i nh ib it io n (% ) Concentration (µg/ml) KB CasKi HCT 116 MCF7 A549 MRC-5

the statement that doxorubicin is a potent cytostatic drug which is applied for the treatment of cancer diseases but the routine use of this drug could bring major adverse effect (Gillle et al., 2002).

Figure 4.26: The in vitro growth inhibitions of selected human cells by doxorubicin as positive reference standard determined by neutral red cytotoxicity assay. Each

value is expressed as mean ± standard deviation of three measurements.

Table 4.16: The IC50 values of doxorubicin against various cancer and non-cancer

cell lines tested

Cell line IC50 values in µg/ml (µM)

KB 0.0125 (0.023) CasKi 0.0060 (0.011) HCT 116 0.3600 (0.663) MCF7 0.0755 (0.139) A549 0.2200 (0.401) MRC-5 0.5500 (1.01)

168 4.4.3 Comparison of cytotoxic activity of P. bleo and P. grandifolia

The results of preliminary cytotoxicity screening of the methanol and fractionated extracts of both Pereskia spp. against various cancer and non-cancer cell lines are summarized in Table 4.17.

The ethyl acetate extracts of both Pereskia spp. in general gave higher inhibition and stimulation values against various cancerous cell lines compared to other extracts. The ethyl acetate extract of P. bleo was selectively toxic against the KB cells. Both ethyl acetate extracts displayed distinctively cytotoxicity effect on KB cell line, which reached IC50 values at relatively low concentration. The ethyl acetate extract of P.

bleo was more active with IC50 value of 4.5 µg/ml against KB cells, in comparison to

16.0 µg/ml shown by that of P. grandifolia. The ethyl acetate extract of P. grandifolia, on the other hand displayed good inhibition against MCF7 cells with IC50 of 20.0 µg/ml.

The active ingredients in the ethyl acetate extract may lead to valuable compounds that may have the ability to kill KB cancer cells but exert no damage to normal cells (IC50 >

100.0 µg/ml against normal cells, MRC-5). The stronger inhibitory effect of the ethyl acetate extracts in comparison to the crude methanol extracts was probably due to the partial purification process whereby the active ingredients are concentrated in the ethyl acetate extract.

Meanwhile, the hexane extract of P. grandifolia also demonstrated remarkable high inhibition towards KB cells with IC50 value of 5 µg/ml, in comparison to the

hexane extract of P. bleo. The water extracts of both Pereskia spp. were found to have no effect on the cancer cell lines (IC50 > 100.0 µg/ml in all cases). All the extracts of

169

A549 cells compared to other cells and no activity against normal cells (IC50 > 100.0

µg/ml).

From Table 4.17, it is observed that the crude methanol extract of P. bleo showed a higher inhibitory effect than the fractionated extracts against the CasKi cells. This might be explained as the compounds that are active against the CasKi cells had decomposed during the fractionation process. In addition, observed activity in the total extracts might be due to synergism between components which were separated as a result of the fractionation process. The synergism among the components in the mixture contributed to the cytotoxic activity, not only dependent on the concentration of certain components, but also on the structure and interaction among the components.

In addition, the cytotoxic effect of the positive reference standards (doxorubicin) were relatively more pronounced than the tested extracts. Although the cytotoxic activities of these extracts are not as effective as doxorubicin, they however have low toxicity against normal MRC5 cell line in comparison to doxorubicin.

In summary, the findings from the cytotoxic activity of both Pereskia spp. extracts support the common belief that ethnopharmacological selection of Pereskia spp. is a useful criterion in drug discovery. It is interesting to note that the extract of the plant showed much less cytotoxicity against the normal cell line, and, if this also occurs in vivo (section 4.7), the use of this plant by locals for the treatment of cancer would have some scientific support. The active extracts were being investigated further to detect the DNA fragmentation (section 4.5) and to determine the active ingredients (section 4.8 and 4.9) present in the extract since these might identify valuable lead compounds in the light of their ability to kill cancer cells but exert little damage as possible on normal cells.

170 Table 4.17: Comparison between IC50 values of P. bleo and P. grandifolia extracts

against various cancer and non-cancer cell lines

Plants / Standard Extracts IC50 (µg/ml) KB CasKi HCT 116 MCF7 A549 MRC-5 P. bleo Methanol 6.5 40.5 41.0 39.0 56 61.0 Hexane 28.0 89.5 67.5 25.0 > 100 > 100.0 Ethyl Acetate 4.5 58.0 22.0 28.0 41 > 100.0 Water > 100.0 > 100.0 > 100.0 > 100.0 > 100 > 100.0 P. grandifolia Methanol 34.0 50.0 53.0 88.0 60 68.5 Hexane 5.0 40.0 25.5 49.0 44 > 100.0 Ethyl Acetate 16.0 21.0 52.0 20.0 45 > 100.0 Water > 100.0 > 100.0 > 100.0 > 100.0 > 100 > 100.0 Doxorubicin (Positive reference standard) 0.0125 0.0060 0.3600 0.0755 0.2200 0.5500 IC50 ≤ 20.0 µg/ml: Active

4.5 Detection of DNA fragmentation

Chromosomal DNA fragmentation at internucleosomal sites is the earliest hallmark of the nuclear events and the most extensively studied biochemical event in apoptosis. Apoptosis has become increasingly important to many areas of biomedical research and internucleosomal DNA fragmentation is frequently used to show the existence of apoptosis (Zhu and Wang, 1997).

In this study, DNA fragmentation was detected using DeadEndTM Colorimetric Apoptosis Detection System as described by the manufacturer (Promega). DeadEndTM