Abstract
Background: The occurrence of cancer diagnosis has become increasingly common and has statistically become one of the most prominent causes of death worldwide. Factors contributing to cancer formation include lifestyle and personal habits, genetics, and external oncogenic stimuli. Cancer occurs through progressive mutation of a cell’s natural genome, enabling the development and survival of the mutated cell. The primary survival mechanism developed is the evasion of the natural apoptotic response; the removal of irregular cells. Pancreatic and colorectal cancers are two of the most lethal cancers. This stems from late diagnosis and poor prognosis due to their asymptomatic nature. Numerous cancer treatments are proven to aid in combatting cancer however, they are not always effective and may produce dangerous side effects. Cancer treatments are also costly to administer and develop. Medicinal plants are seen as an alternative to existing cancer treatments. Many have anti-cancer properties however, the plant metabolites/lead compounds which bring about this activity may not be known or studied. An example of such a plant is Millettia griffoniana. The compounds in medicinal plants are extracted, identified and investigated for their potential anti-cancer activity to identify compounds that can be further studied for development into naturally derived anti-cancer drugs.
Aim and objectives: This study aimed to isolate and identify compounds from M. griffoniana and test their potential anti-cancer activity on MIA PaCa-2 pancreatic cancer, HT-29 colorectal cancer and human embryonic kidney (HEK-293) cells. The objectives included investigating changes in cellular viability using an AlamarBlue assay and a real-time xCELLigence assay and examining the induction of apoptosis, by these compounds, using a Caspase 3/7 assay, light microscopy and Real-Time PCR.
Experimental procedure: The seeds and root bark of M. griffoniana had undergone extraction separately using acetone and chloroform. These crude extracts were then subject to silica-based column chromatography. Compounds from the root bark were isolated directly from the silica column and compounds from the seeds were isolated using Sephadex LH20 after the crude extract was fractioned on the silica column. MIA PaCa-2, HT-29 and HEK-293 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM). The series of isolated compounds were screened using an AlamarBlue assay to identify compounds having cytotoxic activity to allow for further study of their viability as an alternative, naturally derived cancer treatments. Two compounds, compound 5 (durmillone) and compound 7 (griffoniana 55), were selected and griffoniana 55, which had not been identified, underwent 2D NMR and structural elucidation to find the compound identity and structure. Their cytotoxicity against an untreated control, a negative control of 0.1% DMSO and a positive control of 100 μM etoposide
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was assessed through a concentration-series cellular viability assay using AlamarBlue and xCELLigence assays. The IC50s were obtained from the xCELLigence assay and were used to conduct a Caspase 3/7 assay using the Promega Caspase Glo® 3/7 activity assay kit according to the manufacturer’s protocol. The changes in cell morphology of the cancer cell lines treated with the IC50 concentrations of the two compounds were examined using light microscopy. Lastly, to measure the gene expression levels of apoptosis-related genes BAX, BCL-2 and p53, RNA was extracted from cells treated with the IC50s of the two compounds using the Promega ReliaPrep™ RNA Cell Miniprep System. To make it more compatible with PCR amplification, the extracted RNA was reverse transcribed into cDNA which was synthesised using the Promega GoScript™ Reverse Transcription System. The resulting cDNA was subjected to Real-Time PCR using the Biorad iTaq™ Universal SYBR® Green Supermix to quantify the expression of the three genes of interest.
Results and discussion: Seven pure compounds were isolated from M. griffoniana which were screened for cytotoxic effects at a concentration of 100 μg/ml over 24 and 48 hrs. Compounds 5 and 7 were selected for further studies due to their prominent cytotoxic activity. Compound 5 had previously been identified as durmillone and compound 7, given the placeholder name griffoniana 55, had undergone 2D NMR to determine the identity and structure of the compound, which was identified as isojamaicin. Both compounds had varying concentration-dependent cytotoxic activity in the cancer cell lines which was observed in both the AlamarBlue and xCELLigence assays. Minimal cytotoxic activity on the HEK-293 cell line was observed. The IC50 values generated from the xCELLigence analysis were; 32.9 μg/ml for durmillone and 55.46 μg/ml isojamaicin for the MIA PaCa-2 cell line and 25.33 μg/ml durmillone and 39.53 μg/ml isojamaicin for the HT-29 cell line, were used for further studies. Caspase 3 and 7 activity was found to be up-regulated in both MIA PaCa-2 and HT-29 cell line post-treatment with the IC50 concentrations of durmillone and isojamaicin. Visible physical changes associated with apoptosis such as cell shrinkage, and abnormal cell shape and size were observed in both cancer cell lines. Apoptosis induction was investigated by observing the fluctuations in gene expression levels of BAX, BCL-2 and p53. Both the MIA PaCa-2 and HT-29 cell lines were shown to up-regulate BAX and down-regulate BCL-2, which is characteristic of the apoptotic pathway, and this was seen after treatment with the IC50s of durmillone and isojamaicin. p53 was down-regulated in both cell lines which surmised the inability of durmillone and isojamaicin to induce p53-mediated apoptosis.
Conclusion: Durmillone and isojamaicin displayed cytotoxic activity on MIA PaCa-2 and HT-29 cancer cells where the activity of each differed between the two cancer cell lines indicated by the AlamarBlue and xCELLigence assays. Apoptotic activity induced by the compounds was verified due to their up-regulation of caspase activity, evident apoptotic morphological changes, and the up-regulation of BAX together with the down-regulation of BCL-2 and p53.
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Further studies are required to identify the functional differences between these compounds and to examine the mechanisms by which these compounds bring about their cytotoxic activity.
Keywords: anti-cancer M. griffoniana durmillone isojamaicin apoptosis cytotoxic activity.