Integr Cancer Ther. 2013 Sep;12(5):433-41. doi: 10.1177/1534735413485419.

Antihyperglycemic drug Gymnema sylvestre also shows anticancer potentials in human melanoma A375 cells via reactive oxygen species generation and mitochondria-dependent caspase pathway.

Chakraborty D, Ghosh S, Bishayee K, Mukherjee A, Sikdar S, Khuda-Bukhsh AR.

University of Kalyani, Kalyani, India.

 

Abstract

OBJECTIVE: Ethanolic extract of Gymnema sylvestre (GS) leaves is used as a potent antidiabetic drug in various systems of alternative medicine, including homeopathy. The present study was aimed at examining if GS also had anticancer potentials, and if it had, to elucidate its possible mechanism of action.

METHODS: We initially tested possible anticancer potential of GS on A375 cells (human skin melanoma) through MTT assay and determined cytotoxicity levels in A375 and normal liver cells; we then thoroughly studied its apoptotic effects on A375 cells through protocols such as Hoechst 33258, H2DCFDA, and rhodamine 123 staining and conducted ELISA for cytochrome c, caspase 3, and PARP activity levels; we determined the mRNA level expression of cytochrome c, caspase 3, Bcl2, Bax, PARP, ICAD, and EGFR signaling genes through semiquantitative reverse transcriptase polymerase chain reaction and conducted Western blot analysis of caspase 3 and PARP. We also analyzed cell cycle events, determined reactive oxygen species accumulation, measured annexin V-FITC/PI and rhodamine 123 intensity by flow cytometry.

RESULTS: Compared with both normal liver cells and drug-untreated A375, the mortality of GS-treated A375 cells increased in a dose-dependent manner. Additionally, GS induced nuclear DNA fragmentation and showed an increased level of mRNA expression of apoptotic signal related genes cytochrome c, caspase 3, PARP, Bax, and reduced expression level of ICAD, EGFR, and the anti-apoptotic gene Bcl2.

CONCLUSION: Overall results indicate GS to have significant anticancer effect on A375 cells apart from its reported antidiabetic effect, indicating possibility of its palliative use in patients with symptoms of both the diseases.

KEYWORDS: A375 melanoma cells, DNA damage, Gymnema sylvestre, anticancer potential, apoptosis, reactive oxygen species

PMID: 23615751

 

SUPPLEMENTS:

Gymnemasylvestre (GS) is a well-known anti-diabetic drug, used in various systems of alternative medicines, including homeopathy. The anticancer potential of this drug had not been known or evaluated earlier. So this study was designed for examining whether GS could also have capability to induce apoptosis to the cancer cells, signifying its anticancer potential as well. On getting a positive result, the possible mechanism of action of GS against cancer cells was also explored.

For testing anticancer potential, we initially determined cytotoxicity levels of GS in A375 (human skin melanoma) and in normal liver cells (WRL-68).  On being satisfied about its cytotoxic effects in cancer cells, we proceeded to thoroughly analyze its apoptotic effects on A375 cells through certain widely accepted protocols such as Hoechst 33258, H2DCFDA, and rhodamine 123 staining and conducted ELISA for cytochrome c, caspase 3, and PARP activity levels; we also determined the mRNA level expression of cytochrome c, caspase 3, Bcl2, Bax, PARP, ICAD, and EGFR signalling genes through semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and conducted Western blot analysis of caspase 3 and PARP. We also analysed cell cycle events, determined reactive oxygen species accumulation, and measured annexin V-FITC/PI and rhodamine 123 intensity by flow cytometry.

Khuda Bukhsh-fig1Fig 1. Gymnemasylvestre (GS) treatment increased cellular reactive oxygen species (ROS) accumulation (A-H) in A375 cells. Cells were untreated or treated with 200μg/mL for 1, 2, and 3 hours, respectively, and then incubated with H2DCFDA (20μM) for 30 minutes prior to fluorescence microscopic and flow cytometric analysis. (A, B) Control cells. (C, D) GS treated for 1 hour. (E, F): GS treated for 2 hours. (G, H) GS treated for 3 hours. The effects of GS on reduction of ΔΨm (I-P) in A375 cells. Cells were either untreated or treated with 200μg/mL (D2) of GS for 12, 18, and 24 hours, respectively, and were stained with rhodamine 123. After staining, the cells were viewed by fluorescence microscope. (I, J) Control cells. (K, L) GS treated for 12 hours. (M, N) GS treated for 18 hours. (O, P) GS treated for 24 hours.

 

In this study, we observed that whereas GS had little or no cytotoxicity on normal liver cell line WRL-68, it had considerable apoptotic effect on A375 cells. The treatment with GS on A375 cells caused an increase in cellular ROS accumulation at an early hour of drug exposure (within 3 hours). With the ROS accumulation, decrease in mitochondrial membrane potential from 47.0% (in control cells) to 7.9% (in GS-treated cells) after 24 hours of incubation with 200μg/mL of GS treatment, the event towards initiation of apoptosis presumably got triggered. .

Apart from these cytological events, the molecular level analysis revealed the release of cytochrome c after GS treatment.  ELISA and RT-PCR studies also indicated that pro-apoptotic mRNAs of caspase 3, Bax, and PARP genes were up-regulated whereas the anti-apoptotic mRNAs of Bcl2 gene was down-regulated. The altered ratio of the Bax/Bcl2 and the increase in caspase activity possibly led to the apoptosis of the A375 cells. Activated caspase 3 is known to cleave and up-regulate some specific substrates, such as PARP proteins, the activities of which were also detected in the GS-treated cells. The increased expression of PARP supports the DNA breakage in apoptosis. Down-regulation of ICAD indicates that the active CAD molecule can enter the nucleus and degrade the chromosomes. Furthermore, we investigated the effects of GS on the expression of caspase 3 and PARP proteins by ELISA, which confirmed results obtained in RT-PCR studies. GS treatment promoted cytochrome c, caspase 3, and PARP protein expressions in a dose dependent manner in the A375 cells. Decrease in mitochondrial membrane potential possibly induced altered expressions of caspase 3 and PARP proteins that could direct the cells toward apoptosis. Meanwhile the EGFR expression at mRNA level was down-regulated by the GS-treatment, indicating an added anti-proliferative role of GS.

The anti-diabetic property of GS had already been known, but its anticancer potentials had never been explored. This study clearly demonstrates that GS has considerable anticancer potential as well. Furthermore, the possible signal pathway of the drug has also been elucidated for the first time. The apoptotic pathway was linked to the mitochondria-dependent caspase route through caspase 3 and PARP activation, causing death to the cancer cells. Thus this study clearly indicates the possibility of use of GS in formulation of drug that can help patients suffering from a dual attack of diabetes and cancer.

Khuda Bukhsh-fig2Fig2. Flow cytometric analysis. Cells were analyzed after incubation with Gymnema sylvestre (GS) for 24 hours by flow cytometry for annexin V-FITC/PI (A-D) and TUNEL assay (E-H).

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