The use of a 2d cell culture model to investigate the effects of selected plants on tubularization of cervical cancer cell lines

Abstract

Background The current available cell-based in vitro assays for preliminary screening of potential chemotherapeutic agents focus on cell viability and apoptosis of cancer cells. There are no validated high throughput methods that focus on vascularization and tubularization, the two biological processes critical for the process of metastasis of cancer. Malignancy of cancer cells is the cause of mortality in cancer patients. It is projected that halting metastasis in cervical cancer, for example, can reduce the death rates by more than half. The process of metastasis is highly dependent on unregulated over-proliferation, tubularization and vascularization of cancer cells, which all promote localized benign tumors to malignancy. This project investigated tubularization of cervical cancer cell lines, and the anti-tubularization activity of two medicinal plants; Boscia albitrunica and Steganotaenia araliacea hochst, cited to have anti-cancer properties. Without proliferation, differentiation, tubularization and vascularization, both angiogenesis and metastasis cannot occur, which means neoplasms remain localized, and metastasis halted. Methods Phytochemical composition of both plant extracts was investigated. Boscia albitrunica and Steganotaenia araliacea hochst organic and water extracts were investigated for their growth inhibition potential of HeLa and C33A cervical cancer cell lines monolayer using MTT assay. Tubular-like structure formation ability of cells was investigated through 2-D cell culture model using Matrigel to investigate the most active fraction extract. The 2-D Matrigel technology was employed to grow cells on and through the Matrigel. Long distance objective light microscope was used to view the formation or non-formation of cellular tubular-like structures after cancer cells were treated with plant extracts that showed highest anti-proliferation activity on monolayer cells. Results Phytochemical analysis of raw plant materials of both plants showed the presence of secondary metabolites including alkaloids and flavonoids, known to possess various pharmacological activities including anticancer activity. Steganotaenia araliacea hochst and Boscia albitrunca showed the IC50 on both cell lines. B. albitrunca MeOH extract displayed a high IC50 value of 28.6931 μg/ml. This value indicated very low hazardous cytotoxicity status. S. araliacea hochst MeOH extract displayed an IC50 value of 14.78 μg/ml and indicated a moderate hazard and potency of cytotoxicity. Boscia albitrunica methanol and DCM extracts exhibited below 42% and 30% cell growth inhibition against HeLa and C33A cell lines respectively. Steganotaenia araliacea hochst water extract showed over 60% growth inhibition at 10 μg/ml concentration on HeLa cell line and was further fractionated by solid phase extraction (SPE) to concentrate the active constituents. Fraction 87 of the extract showed the most cytotoxic activity on HeLa cell line and was used to investigate vascularization and tubularization on the 2-D Matrigel cell culture model using the HeLa cell line. The 87 SPE fraction of S. araliacea hochst water extract displayed anti-tubularization mechanisms in the 2-D Matrigel cell culture system at 10 ug/ml in positive (Vincristine), negative control (untreated) and treated wells individually. Conclusion The advanced 2-D Matrigel sandwich cell culture model used in this study can be used for high throughput screening for potential anti-metastasis, anti-tubularization agents.