The effect of piperine on cell invasion and metastasis

Similar to the approach of Kuttan et al. (2004), Y.P. Hwang et al (2011) carried out his investigation on piperine, even though not on the same research line of Kuttan et al (2004), at the molecular level, involving matrix metalloproteases (MMPs) which play a vital role in the tissue invasion, angiogenesis, inflammatory tissue destruction and cancer cell metastasis. They investigated the inhibitory effects of piperine on tumor invasion and migration and the possible mechanisms involved using human fibrosarcoma HT-1080 cells and also found that piperine suppresses PMA-enhanced matrix metalloproteinase-9 (MMP-9) expression at the protein, mRNA, and transcriptional levels through the suppression of NF-kB and AP-1 activation without changing the level of tissue inhibitor of metalloproteinase (TIMP)-1. Before exploring the pharmacological effect of piperine on PMA induced MMP activity, toxicity studies were carried out and confirmed that dose less than 200??M is considered to be safe and had no cytotoxicity. In order to find out the effect of piperine on cell invasion and metastasis, in-vitro assays such as transwell and wound healing assays were carried out. As per the results of these assays, it was found that piperine inhibited the HT-1080 cell migration. In addition to the above assays, matrigel invasion assay was also carried out which showed that the PMA induced cell invasion was reduced with the treatment of piperine. Additionally, gelatin zymography and western blotting techniques were done to investigate the effect of piperine on the inhibition of PMA induced MMP activity which showed that piperine inhibited PMA induced MMP-9 activity. To find out whether the decresed MMP-9 activity is by reduced transcription, RT-PCR was done. The results of RT-PCR showed that there is a decreased transcription of MMP-9.
Among the characteristics of cancers, cell proliferation i.e. enabling the limitless replicative potential of the cancer cell is one of the significant characteristics. Wan Lee et al. (2013) assessed antiproliferative effect of piperine on human prostate cancer PC-3 cells. In order to determine the effect of piperine as anti proliferative MMT assay was performed. The results showed that there was a reduction in the cell viability. It was found to inhibit the growth of androgen independent PC-3 and androgen dependent LNCaP prostate cells in a dose-dependent (30’90 ??M) and time-dependent (24’48 h) manner. According to the data collected, the inhibitory concentration IC50 value was also estimated as 40 ??M. He also performed flow cytometry in order to investigate that piperine causes cell cycle arrest in G0/G1 phase and build up of sub G1 population in PC-3 cells. The data obtained from flow cytometry indicated that due to the blockade of G0/G1 phase as well as by the induction of apoptosis by piperine, the growth of the PC-3 cells was inhibited. Moreover, Wan Lee et al. also checked the levels of cell cycle progression related molecules, cyclins and CDKs, using western blotting. The results showed that the levels of cyclin D1, cyclin E, CDK2 and CDK4 were decreased by piperine. It was also found by the phase contrast microscope that piperine treatment changes the cytomorphology of the PC-3 cells from adherent, homogenous to non-adherent, detached, round structure. The nuclear changes were also found to alter by piperine treatment which showed apoptotic features compared to the untreated cells. These morphological changes indicate the piperine treatment can cause the reduction in the number of PC-3 cells by inducing cell death through apoptosis. For apoptosis to take place, caspase 3 has to be activated and break down of a protein PARP [ploy-ADP ribose- polymerase] shouls take place. Wan Lee et al. investigated that piperine was involved in carrying out the activation of caspase-3 and also breaking down PARP which was also confirmed by using a colorimetric assay. Along with above tests, DCHF-DA fluorescence intensity test was performed to investigate if the production of ROS was involved in the induction of apoptosis by piperine. The results of the test showed that production of ROS was vital in the piperine induced apoptosis of PC-3 cells.
On the other hand, Minh Truong Do et al. (2013) and Paul B Yaffe et al. (2013) investigated the effect of piperine to induce apoptosis in the cancer cells. Minh Truong Do et al. (2013) found that piperine exhibits anti-tumor effects in HER2 over expressing breast cancer cells. It exerted anti-tumor activity by inhibiting proliferation and inducing apoptosis through caspase-3 activation and PARP cleavage and it also inhibited HER2 gene expression at the transcriptional level. MMT assay was performed to investigate the effect of piperine on the growth of HER2- over expressing breast cancer cells. The results of the assay showed that piperine decreased the growth of the HER2 expressing SKBR3cells in dose dependent manner. Taking PARP expression and caspase-3 activity as markers for apoptosis, an assay was performed to find out the piperine induced caspase-3 activity which showed that caspase-3 was activated and also PARP protein was broken down which are the key steps in the apoptosis. In addition to the above assay, western blotting and qPCR was also performed to investigate whether piperine decreases HER2 expression in breast cancer cells. The results have showed that piperine treatment (10-50 ??M) for 48 hrs on SKBR3 cells showed the reduction in the HER2 protein and mRNA levels. Again, to find the effect of piperine on ERK 1/2 signalling, which interferes the transcriptional activity of SREBPs, western blotting was performed. The results showed that piperine the decreased the phosphorylation of ERK 1/2. It was also found by quantitative RT-PCR that piperine decreased the SREBP and FAS mRNA levels. Additionally, anti-metastatic activity of piperine was also investigated by wound healing assay, which showed that piperine at the doses 25 and 50 ??M decreased the migration of HER3 over expressing breast cancer cells.
Similarly, Paul B Yaffe et al. (2012) investigated the effects of piperine in rectal adenocarcinoma cells to cause the impairment of cell cycle progression and induce apoptosis through reactive oxygen species (ROS). They investigated the effect of piperine on the growth of HRT-18 human rectal adenocarcinoma cells using MTT assay which showed that the metabolic activity of HRT-18 cells was inhibited by piperine in a dose- and time-dependent fashion. This activity indicates that piperine has cytostatic/cytotoxic activity. In order to investigate the inhibitory effect of piperine on cell cycle progression, flow cytometric analysis of Oregon Green 488-stained was carried out at the dose of 150 ??M piperine for 72 hrs which caused an increase in the HRT-18 cells in G0/G2 phase and decrease in the percentage of cells in G2/M phase. To find whether piperine caused apoptosis or necrosis of HRT-18 cells to die, Annexin-V-FLUOS staining and PI staining was performed. The phase- contrast photo micrographs of HRT-18 cells showed that the number of cells were decreased and found to have a apoptotic cell morphology. As the increased ROS induces apoptosis, Paul B Yaffe et al. used flow cytometric analysis of dihydroethidium- and 2′,7′-dichlorofluorescein diacetate-stained HRT-18 cells to find whether piperine was involved in the production of ROS. The results showed that piperine treated cells showed minimum production of superoxide anion and relatively higher production of hydroxyl radical production.
In the present review of literature, the contribution of various researchers suggest that piperine has anti ‘ cancer properties aiming at the characteristics of the cancer cells like metastasis and tissue invasion, escaping apoptosis and ability to proliferate. In addition to these properties piperine was also found to ameliorate the effect of carcinogens and also can annul multidrug resistance. Therefore, considering the above facts piperine can be considered as a potential treatment for cancer.

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