Evaluation of Anti-Proliferative Action of a Poly Herbal Formulae ‘Le Pana Guliya’ and Assessment of Its Mechanism of Action

Abstract

‘Le Pana Guliya’ (LPG) is a polyherbal formulation which is used to treat different types of cancers in traditional medicine of Sri Lanka. The present study describes in vitro efficacy and biochemical mechanism of action of LPG on HepG2, HeLa, MCF-7and RD cell line compared to a healthy CC1 cells. MTT, LDH and protein content assays were carried out to evaluate the antiproliferative activity of LPG on tested cancer cells compared to the CC1 cell line. NO synthesis and GSH content were assayed to determine the oxidative stress exerted by LPG. Rhodamine 123 staining, caspase 3 activity, DNA fragmentation and microscopic examination of cells stained with EtBr/AO were used to identify the apoptosis mechanisms associated with LPG. The MTT assay demonstrated significant antiproliferative activity (p<0.05) against HepG2, HeLa, MCF-7 and RD cells after 24 hour exposure of LPG compared to CC1 cells and EC50 values were 2.7 ± 0.4, 19.0 ± 2.6, 24.9 ± 2.0 and 17.6 ± 2.8 μg/mL respectively. In contrast, CC1 cells showed an EC50 value of 213.07 ± 7.71 μg/mL. Similar results were observed with LDH release where there was a significant (p<0.05) dose and time dependent increase in the LDH release in cancer cells. A dose dependent decrease in protein content was shown in all cancer cell types compared to respective untreated cells after 24 hour exposure. However the decrease in protein content of CC1 cells treated with LPG (2.5 -10 μg/mL) was lower than that of all cancer cell types and the values were less than 20%. There was a significant increase in NO production and decrease in GSH production (p < 0.05) in all cancer cell types treated with LPG compared to their respective untreated cells as well as with CC1 cells. The exogenous GSH was able to increase the cell viability significantly (p < 0.05) in the presence of LPG in all cell types investigated. Rhodamine 123 assay showed the mitochondrial involvement in cell death by depletion of MMP. The decreased MMP in turn led to the increase in caspase-3 activity. Thus a prominent increase in caspase 3 activity was observed in HepG2, HeLa and RD cells between the concentrations of 0.25 and 25 μg/mL. The MCF-7 and CC1 cells did not show an elevation in caspase 3 activity. HepG2, HeLa and RD cells showed typical apoptotic morphological changes and fragmented DNA in contrast to the MCF-7 cells and in CC1 cells even at high concentrations of LPG (500 µg/mL). The present study proves that LPG is able to induce apoptosis via oxidative stress mediated caspase 3 dependent pathway in HepG2, HeLa and RD cells. Even though the LPG exerts oxidative stress in MCF-7 cells by elevating the NO levels and decreasing the GSH levels, the absence of typical apoptotic features indicate that MCF-7 cells follow an alternative cell death pathway. Results of brine shrimp and CC1 cells prove that LPG does not exert cytotoxicity on healthy cells.