The results suggest that VA induced a strong cytotoxic effect on MCF-7 and MDA-MB-231 cells by blocking their proliferation, with IC50 values of ≤100 µg/ml, respectively. The IC50 values of water-soluble VA extract in MCF-7 cells reported in other studies demonstrate enormous discrepancy, ranging from 5.6 µg/ml to 1000 µg/ml [2], [15], [16]. As plant extract comprises several different active biological compounds, it is likely that the inconsistency of VA potencies is due to batch variation, in which certain batches may have higher activity than others. Despite the inconsistency of IC50 values, the present study nevertheless confirms the growth inhibitory effect of VA in MCF-7 cells. It was also showed, for the first time, that VA demonstrated its ability to inhibit proliferation of MDA-MB-231 cells at a potency that is slightly lower than that in MCF-7 cells. These results suggest that VA is able to inhibit the proliferation of human breast cancer cells regardless of the cell type, highlighting its broad-spectrum cytotoxic effects.
Despite the fact that VA inhibited growth of both MCF-7 and MDA-MB-231 cells, it blocked proliferation by inducing cell cycle arrest at G1/S phase in MCF-7, but not in MDA-MB-231 cells. This selective effect of VA thus prompted an analysis of the differential changes in cell cycle regulatory proteins induced by VA. Western blot analysis has revealed that VA induced activation of p53 and p21 while suppressing the expression of cyclin D1 and cyclin E in MCF-7 cells. This provides strong evidence that VA arrested MCF-7 cells in the G1 phase, thereby inhibiting their progression to the S phase. However, there were no significant changes in the expression of p53, p21, cyclin D1 and cyclin E after VA treatment in MDA-MB-231 cells. Owing to this observation, this study made the postulation that this selective effect of VA on cell cycle arrest in MCF-7 cells could be due to the difference in p53 status between the two cell lines. Unexpectedly, the partial suppression of p53 transcriptional activity by PFT-α failed to reverse the cell cycle arrest induced by VA in the MCF-7 cells as shown in Figure 8. Thus, it is possible that the VA-induced cell cycle arrest in MCF-7 cells may be independent of p53.
Although p53 may not responsible for the VA-induced cell cycle arrest in our study, the increased expression as detected by western blotting is believed to be due to the DNA damage induced by VA. This postulation can be supported by a report in which VA was shown to induce minimal DNA damage in MCF-7 cells as assessed by alkaline single cell gel electrophoresis (Comet) assay [2]. Since it is widely agreed that DNA-damage response is integral to the actions of p53 as a tumour suppressor, it is believed that VA can induce DNA damage in MCF-7 cells, which in turn activates p53 due to the release from Mdm2, and subsequently triggers various downstream effects.
The results from this study indicated that VA induced apoptosis in MCF-7 and MDA-MB-231 cells as measured by flow cytometry. At the molecular level, VA treatment down-regulated the expression of Bcl-2 and Bcl-xL and up-regulated the expression of Bax and Bak in MCF-7 and MDA-MB-231 cells. This suggests the inhibition of the anti-apoptotic signal and the induction of the pro-apoptotic signal, thereby affecting the mitochondrial permeability. Given that the results showed the activation of caspase-9, it is possible that VA stimulated the release of cytochrome c, which in turn facilitated the formation of apoptosome complexes. The results also showed the activation of caspase-3 and caspase-7 in MDA-MB-231 cells, and caspase-7 in MCF-7 cells, after which both were followed by PARP cleavage. One possible mechanism by which VA induces apoptosis is thus through modulating the expression of Bcl-2 family members to affect membrane permeability, which in turn results in the sequential activation of caspase-9, caspase-3 and/or -7 and ultimately, PARP cleavage. These observations are in agreement with a report in which VA was shown to alter the cell membrane permeability in MCF-7 cells [16]. The results also indicate that VA induces apoptosis partly through the activation of caspase-8 in MCF-7 and MDA-MB-231 cells. However, the mechanism by which VA activates caspase-8 remains to be elucidated. Nevertheless, this study suggests another possible mode of action by which VA induces apoptosis through the induction of the extrinsic apoptotic pathway. It is still unclear whether VA-induced activation of caspase-8 can cleave Bid, which is a BH3-only pro-apoptotic protein that can initiate the mitochondrial pathway. Further research on the expression of Bid is required to determine if cross-talk between the mitochondrial intrinsic pathway and the death-receptor-mediated extrinsic pathway does exist in VA-induced apoptosis.
Although caspase may be a necessary factor in the execution of programmed cell death, the process of caspase activation is not the sole factor in determining the triggering of apoptosis. Some studies have reported the model of caspase-independent cell death in different cell types, such as Jurkat, MCF-7 and Hela cells [17]-[19]. Therefore, it is important to determine if VA-induced apoptosis can still occur in the presence of the caspase inhibitor, z-VAD-fmk. Z-VAD-fmk is a cell-permeable tripeptide inhibitor which contains aspartate residue and fmk group, mimicking the cleavage site of caspase and forming a covalent inhibitor/enzyme complex [20]. It works by binding irreversibly to the catalytic site of caspases [20]. The results from this study highlight the important role of caspases in VA-induced apoptosis in MCF-7 cells because the inhibition of caspase activity by z-VAD-fmk abolished the PARP cleavage, suppressing the overall apoptosis induced by VA. It is thus possible that VA inhibits the growth of MCF-7 cells through the induction of caspase-dependent apoptosis.
By contrast, the results showed that VA-induced apoptosis in MCF-7 cells was not mediated through a p53-dependent pathway. For the past three decades, p53 has been the subject of intense research interest. The p53 tumour suppressor has been termed ‘the guardian of the genome’ because of its pivotal role in safeguarding the integrity of genetic information in response to various genotoxic injuries [21], [22]. Besides the previously mentioned role of suppressing growth arrest, the induction of apoptosis is one of the central activities by which p53 exerts its tumour-suppressing function. It has been widely known that p53, as a transcription factor, promotes apoptosis through the transcription of its target genes such as Bcl-2 family members. However, an increasing number of studies has shown the existence of a transcription-independent mechanism – i.e. a direct localization of p53 to the mitochondria, such that p53 can interact directly with Bcl-2 or Bcl-xL to promote apoptosis [22], [23]. Thus, it is of paramount importance to determine whether p53 plays a p53 transcription-dependent or independent role in the VA-induced apoptosis. The results obtained from the study showed that blocking the p53 transcriptional activity in MCF-7 cells by PFT-α could not reverse the VA-induced apoptosis as indicated by Annexin V-FITC/PI assay and western blot analysis. It is likely that VA-induced apoptosis in MCF-7 cells may not occur via a p53 transcription-dependent mechanism. However, further research is required to determine whether it is a p53 transcription-independent mechanism or a pathway that is independent of p53 altogether, by using pifithrin-mu (PFT-μ) in addition to PFT-α. PFT-μ works by reducing the binding affinity of p53 to Bcl-2 and Bcl-xL, thereby inhibiting its binding to mitochondria, but without affecting its transactivation activity [12]. It can help to determine if the apoptosis is independent of p53 transcriptional activity. If both PFT-α and PFT-μ fail to reverse the VA-induced apoptosis, it would mean that the VA-induced apoptosis is independent of p53 altogether.
Hormone receptor status has been recognised as the most important prognostic and predictive factor for response to hormonal therapy [24]. As ER status is used as a determinant factor for the current breast cancer treatment [14], agents that can compromise ER signalling promise to be clinically important therapeutic drugs. ER-α is one of the isoforms of ER, which acts as a transcription factor to initiate the transcription of specific target genes upon activation by estrogen [25]. A crucial finding from this study was that besides modulating the expression of apoptosis-regulating molecules, VA also mediates its effects through down-regulation of ER-α expression in MCF-7 cells. Since around 70% of diagnosed breast cancers are ER-positive which express ER-α in particular, the ability of VA to inhibit ER-α expression suggests the potential clinical significance of VA. Although it is widely known that MDA-MB-231 is an ER-negative breast cancer cell line, western blot analysis has revealed a basal level of ER expression. This observation can be supported by the studies conducted by Ford et al. [26] who showed that both MCF-7 and MDA-MB-231 cell lines express ER-α and ER-β using flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis [26]. Despite the low expression of ER-α in MDA-MB-231 cells, VA was able to reduce its expression, suggesting its high sensitivity towards ER-α. These results provide the basis for future research to further elucidate the potential application of VA as an anti-estrogenic therapeutic agent.
One of the downstream signalling pathways of ER is Akt, which is the master regulator of cell growth and is closely linked to cell survival [27], [28]. The results from this study demonstrate the ability of VA to inhibit the phosphorylation of Akt at Threonine 308 residue, implying the suppression of cell survival and proliferation signals in both MCF-7 and MDA-MB-231 cells. Inhibition of Akt activation is associated with a pro-apoptotic effect by induction of the Bad pro-apoptotic proteins of the Bcl-2 family, leading to apoptosis [28]. Furthermore, Akt inhibition led to the suppression of phosphorylation of GSK3β, which targets cyclin D1 for proteasomal degradation, resulting in cell cycle arrest in the G1 phase [27]. Hence, Akt signalling pathway may be one of the mechanisms of VA in the induction of cell cycle arrest and apoptosis. Other upstream kinases such as PDK1 and PI3K, as well as downstream proteins like JNK, are to be further studied in order to elucidate the actions of VA fully.
There has been growing interest in combination therapy as it induces a greater effect in the improvement of patients' survival [29]. Since cancer is the result of the accumulation of numerous mutations, it is rational to combine two or more drugs with different mechanisms of action to increase cell killing. VA was thus combined with a current chemotherapeutic alkylating agent, doxorubicin, to determine their synergistic effect in human breast cancer cells. Doxorubicin exerts its effects by intercalating base pairs between DNA, thereby inhibiting both DNA and RNA synthesis. In addition, it mediates its main cytotoxic action through inhibiting the activity of topoisomerase II, which is an enzyme responsible for the uncoiling of DNA [30]. These two different mechanisms of action result in DNA disruption that eventually leads to cell death. However, it has been found to be associated with adverse events such as increased risk of bleeding and infection, loss of appetite, cardiac damage and heart failure. In this study, a synergistic effect was observed when VA was combined with doxorubicin in both MCF and MDA-MB-231 cells. This suggests that the combination of VA and doxorubicin at certain concentrations may ameliorate the side effects of doxorubicin treatment. Importantly, the effect of doxorubicin is cell cycle non-specific while that of VA is believed to be G1/S phase-specific. This difference in the mechanism of action allows for more attacks at multiple phases of the cell cycle to accelerate the treatment process, possibly preventing resistance from occurring. Therefore, the synergistic effect was more significant in MCF-7 than MDA-MB-231 cells because VA induces G1/S cell cycle arrest only in MCF-7 cells, which results in more attacks as compared to that of MDA-MB-231 cells. Moreover, different mechanisms of action limit the overlapping toxicities, improving the results of the overall treatment. Hence, these results suggest that VA can act as a complement to current treatment. It is therefore worthwhile to further examine the mechanisms of this synergism so as to evaluate the reasonable applications of VA in human breast cancer treatment.
Conclusion
In conclusion, VA shows anti-cancer effects in MCF-7 and MDA-MB-231 cells. The effect was mediated through the inhibition of cell proliferation of the breast cancer cells. A novel finding was that the underlying mechanisms of this growth inhibition induced by VA involved the suppression of ER-α and the phosphorylation of Akt, stimulation of cell-specific G1/S cell cycle arrest and the induction of apoptosis through both extrinsic and intrinsic apoptotic pathways. In addition, the VA-induced apoptosis in MCF-7 cells is likely to be caspase-dependent and not p53 transcription-dependent, while the cell cycle arrest is independent of p53. VA also exhibited synergism when combined with doxorubicin, suggesting that it can complement current chemotherapeutic treatment. By detailing the complex mechanisms involved in breast cancer cells, this study confirms the hypothesis, demonstrating the potential applications of VA as an anti-cancer drug and thus paving the way for further research on VA in the field of anti-cancer drug discovery.
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