This study examined the cell growth arrest, anti-proliferation, anti-migration, and pro-apoptosis properties of Pluchea indica leaf and root crude aqueous extracts in human brain malignant glioma cell line GBM8401, and human cervical carcinoma cell line HeLa. The underlying inhibitory mechanism of P. indica leaf and root crude aqueous extract was also investigated. It was detected that membrane lipid oxidation level in HeLa cells increased after treatment with P. indica crude aqueous extract. The data showed that P. indica leaf and root crude aqueous extracts were effective in suppressing the proliferation and migration of GBM8401 and HeLa cells. It was found that P. indica root crude aqueous extract inhibited focus formation of GBM8401 and HeLa cells, and promoted apoptosis in HeLa cells. The data indicated that phosphorylated-p53 and p21 were induced in P. indica root crude aqueous extract-treated GBM8401 and HeLa cells, and that phosphorylated-AKT was decreased in HeLa cells treated with P. indica root crude aqueous extract.
The medicinal properties of P. indica extracts have been studied previously. The methanol fraction of P. indica root extract was found to possess antioxidant activity . The methanolic fraction of a chloroform extract of P. indica root was found to have anti-inflammatory activity . Beta-sitosterol and stigmasterol isolated from P. indica root methanol extract was found to be able to neutralize viper and cobra venom and antagonize cobra venom-induced lethality, cardiotoxicity, neurotoxicity, and respiratory changes . In contrast to previous studies, this is the first study to examine the effect of crude aqueous extracts of P. indica leaf and root on cancer cells. Tannins, saponins, flavonoids, phenols, and proanthocyanidins were detected in crude aqueous extracts of P. indica leaf and root, which showed that P. indica has reasonable anti-cancer potential because total phenolics, flavonoids, and tannins were shown to inhibit ATP-binding cassette transports in cancer cells , flavonoid intake was found to associate with a significant reduction in the risk of gastric cancer in women , saponins from Radix astragali were found to suppress colon cancer cell carcinogenic activity by reducing vascular endothelial growth factor , and proanthocyanidins from grape seeds inhibited pancreatic cancer cell growth and induced apoptosis .
Research into the anti-cancer potential of herbal extracts have been growing and expanding [6, 25]. The compound, n-butylidenephthalide, isolated from the chloroform extract of Angelica siensis was found to upregulate the expression of p21 and p27 and increase apoptosis-associated proteins in DBTRG-05MG and RG2 cells and suppress the growth of subcutaneous rat and human brain tumors . Nexrutine, a Phellodendron amurense bark methanol extract, was found to inhibit prostate cancer cell proliferation through modulation of AKT and cAMP-responsive element binding protein (CREB)-mediated signaling pathway, and that Nexrutine activates cyclin D1, which prevents the progression of prostate cancer . In this study, it was observed that P. indica leaf and root aqueous extracts inhibited GBM8401 malignant glioma cells and HeLa cervical carcinoma cell growth, and migration. We found that the crude aqueous extract P. indica root suppressed focus formation of GBM8401 and HeLa cells.
The p53 tumor-suppressor gene does not function properly in most human cancers . In brain cancers, p53 can be inactivated through amino acid-changing mutation in the DNA-binding domain and/or deletion of the p14ARF gene, and in cervical cancers, p53 is inactivated through viral infection [29, 30]. Phosphorylation of p53 by kinases, caused by DNA damage, leads to p53 activation . Phosphorylation of p53 can also occur in response to oxidative stress through the platelet-derived growth factor β receptor (PDGFβ)-mediated ataxia telangiectasia mutated (ATM) kinase activation or direct ATM activation by oxidative stress [32, 33]. One of the first consequences of p53 activation is cell cycle arrest through the p53-dependent expression of p21WAF1/CIP1, an inhibitor of cyclin-dependent kinases (CDKs) . Upregulation of p53 and p21 were found to be induced by lipid peroxidation in previous studies: lipid peroxidation increase was associated with p53 mRNA increase in a rat model , lipid peroxidation product from increased ferrous iron level in lysosomal compartment triggered upregulation of p53 , lipid peroxidation product sensitizes cells to UV-induced killing by inhibiting nucleotide excision repair and forming a peroxide-DNA adduct at codon 249 of the p53 gene , and that hydrogen peroxide-induced lipid peroxide production increased p21 expression . In the present study, it was observed that P. indica root crude aqueous extracts caused an increase in membrane lipid oxidation, and induced phosphorylated-p53 and p21expression in GBM8401 and HeLa cells.
The AKT/PKB (protein kinase B) kinases play important roles in signaling pathways that regulate cellular processes controlling cell proliferation, survival, and genome stability . Hyperactivation of the AKT pathway was implicated in many types of human cancer and dominantly inherited cancer syndrome. AKT phosphorylates and inactivates the pro-apoptotic factors BAD and procaspase-9 . In a pro-cell cycle progression mechanism involving p53, AKT promotes the phosphorylation and translocation of Mdm2 into the nucleus, where it downregulates p53, which antagonizes p53-mediated cell cycle checkpoints . AKT directly antagonizes the function of the cell cycle inhibitors p21WAF1 and p27Kip1 by phosphorylating a site located near the nuclear localization signal to induce cytoplasmic retention of these cell cycle inhibitors . Some investigations have shown elevated AKT activity to be highly prevalent in high grade, late stage and/or metastatic tumors, and several reports have linked AKT activation with reduced patient survival or tumor radio-resistance [42, 43]. In the present study, P. indica root crude aqueous extract treatment lowered the expression of activated phosphorylated-AKT, without affecting the expression of non-phosphorylated AKT, and induced the expression of phosphorylated-p53 and p21 in HeLa cells. Therefore, the pro-apoptotic and anti-proliferation properties of the crude aqueous extract of P. indica root might be attributed to its induction of phosphorylated-p53 and p21 through the downregulation of activated phosphorylated-AKT, which causes cell cycle arrest and initiation of apoptosis leading to cancer cell death.
The active components in crude aqueous extracts of P. indica root may be good candidates for further investigation into the search for effective anti-cancer and synergistic drugs. It is also important to note that aqueous extracts of herbal substance was previously found to be less toxic than ethanol extracts of herbal material; the LD50 of aqueous and ethanol extracts were 12.30 g/kg and 6.15 g/kg, respectively . We found that P. indica leaf and root crude aqueous extracts possess pro-oxidant, anti-proliferation, and anti-migration properties in GBM8401 malignant glioma cells and HeLa cervical carcinoma cells, and that P. indica root crude aqueous extract inhibited focus formation of these cancer cells. We also found that phosphorylated-p53 and p21 are induced in GBM8401 and HeLa cells after P. indica root crude aqueous extracts treatments. As keenly pointed out by a review, the challenge in CAM is to avoid contaminated products and those that may interact with prescription pharmaceuticals . Further examination into the underlying anti-cancer mechanism and pharmacodynamic and pharmacokinetic interactions with current cancer drugs should be carried out to ensure effective clinical application and usage of P. indica leaf or root crude aqueous extract.