Humic acid induces G1 phase arrest and apoptosis in cultured vascular smooth muscle cells

Humic acid (HA) in well water used by the inhabitants for drinking is one of the possible etiological factors for Blackfoot disease (BFD). In this study, the ability of HA to inhibit cell cycle progression and induce apoptosis in cultured smooth muscle cells (SMCs; A7r5) was investigated. Treatment of the SMCs at various HA concentrations (25-200 microg/mL) resulted in sequences of events marked by apoptosis, as shown by loss of cell viability, morphology change, and internucleosomal DNA fragmentation. HA-induced apoptotic cell death that is associated with loss of mitochondrial membrane potential (Delta Psi m), cytochrome c translocation, caspase-3, -8, and -9 activation, poly ADP-ribose polymerase (PARP) degradation, dysregulation of Bcl-2 and Bax, and upregulation of p53 and phospholyrated p53 (p-p53) in SMCs. Flow cytometry analysis demonstrated that HA blocked cell cycle progress in the G1 phase in SMCs. This blockade of cell cycle was associated with reduced amounts of cyclin D1, CDK4, cyclin E, CDK2, and hyperphosphorylated retinoblastoma protein (pRb) in a time-dependent manner. Apparent DNA strand breaks (DNA damage) were also detected in a dose-dependent manner using Single-cell gel electrophoresis assay (comet assay). Furthermore, HA induced dose-dependent elevation of reactive oxygen species (ROS) level in SMCs, and antioxidant vitamin C and Trolox effectively suppressed HA-induced DNA damage and dysregulation of Bcl-2/Bax. Our findings suggest that HA-induced DNA damage, cell cycle arrest, and apoptosis in SMCs may be an underlying mechanisms for the atherosclerosis and thrombosis observed in the BFD endemic region.     

Tebufenozide induces G1/S cell cycle arrest and apoptosis in human cells

Tebufenozide is a non-steroidal insect growth regulator and is extensively used to control pests, although it is considered to be safe for mammals and environmentally friendly. However, previous studies have found that tebufenozide is cytotoxic to man, although the exact mechanism remains elusive. This study will investigate the apoptotic molecular mechanisms which result from tebufenozide-induced DNA damage in HeLa cells. Our results demonstrate that tebufenozide could trigger arrest in G1/S phase related to a downregulation of cyclin E and cyclin-dependent kinase (CDK) 2 protein. In addition, Western blotting showed apoptosis was associated with the upregulation of p53, Bax and cleaved-PARP, as well as downregulation of Bcl-2 and PARP. Tebufenozide also regulated changes in mitochondrial permeability and reduced mitochondrial number and intracellular ATP production. Briefly, these results suggest that tebufenozide- induces cell cycle arrest and apoptosis through activating p53 protein in a Bax- and Bcl-2-triggered mitochondrial pathway. This work provides some scientific context for the safe use of tebufenozide in agriculture.     

Activation of ATM-Chk2 by 16-dehydropregnenolone induces G1 phase arrest and apoptosis in HeLa cells

The natural pregnane steroid 16-dehydropregnenolone (16-DHP) showed the growth inhibitory activity against human tumor cells; however, the mechanisms of actions of 16-DHP were not clarified. In this study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to investigate the growth inhibitory effect of 16-DHP. Cell cycle distribution was analyzed using flow cytometry. Hoechst 33258 staining and DNA agarose gel electrophoresis were used to detect apoptosis. The levels of proteins were probed by Western blotting, and caspase activities were analyzed using Caspase Activity Kit. We found that 16-DHP inhibited the growth of human cervical carcinoma cells (HeLa cells) in a time- and dose-dependent manner. The growth inhibitory effect of 16-DHP was associated with G1 arrest mediated by ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2)-p53 signaling, as demonstrated by induction of the phosphorylations of ATM, Chk2, and p53 proteins. Followed by G1 arrest, 16-DHP-treated HeLa cells underwent caspase-dependent apoptosis. The inhibitors of caspase-3 and caspase-9 but not caspase-8 inhibitor blocked 16-DHP-induced apoptosis. Moreover, 16-DHP increased the level of Bax protein and the release of cytochrome c from mitochondria, but had no effect on the level of Bcl-2. These results suggested that 16-DHP inhibited the growth of HeLa cells via inducing ATM-Chk2-p53 activation-mediated G1 arrest and mitochondrial cell apoptosis.     

Berberine induces G1 arrest and apoptosis in human glioblastoma T98G cells through mitochondrial/caspases pathway

Berberine is an isoquinoline plant alkaloid with a long history of being used for the treatment of many diseases in Chinese herbal medicine. Berberine has a wide range of biochemical and pharmacological effects, including antitumor activities, but its mechanism of action is not clearly understood. In this study, we investigated that the relationship between the antiproliferative activities of berberine and the apoptotic pathway associated with its molecular mechanism of action in human glioblastoma T98G cells. Berberine treatment of T98G cell lines inhibited cell proliferation and induced cell death in a dose (50-200 microg/ml) dependent manner with an IC50 value of 134 microg/ml, which was associated with an increase in G1 arrest. Western blot analysis showed that the berberine-induced G1 arrest was mediated through the increased expression of P27 and the decreased expression of cyclin-dependent kinase (CDK) 2, CDK4, cyclin D, and cyclin E proteins. Berberine treatment also markedly enhanced apoptosis in T98G cells through the induction of a higher ratio of the Bax/Bcl-2 proteins, the disruption of mitochondrial membrane potential, and the activation of procaspase-9, caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP). Berberine can inhibit T98G cell proliferation by inducing G1 arrest and apoptosis. These results demonstrate that the berberine-induced apoptosis of T98G cells is primarily mediated through the mitochondrial/caspases-dependent pathway.     

Artesunate induces G0/G1 cell cycle arrest and iron-mediated mitochondrial apoptosis in A431 human epidermoid carcinoma cells

The anticancer effects of artesunate (ART) have been well documented. However, its potential against skin cancer has not been explored yet. Herein we reported that 60 μmol/l ART effectively inhibited A431 (human epidermoid carcinoma cells) growth but not that of HaCaT (normal human keratinocyte cells). Our results revealed that ART induced cell cycle arrest at G0/G1 phase through the downregulation of cyclin A1, cyclin B, cyclin D1, Cdk2, Cdk4, and Cdk6. This correlated with the upregulation of p21 and p27. The 5-bromodeoxyuridine incorporation assay also indicated that ART treatment reduced DNA synthesis in a time-dependent manner. Furthermore, ART induced mitochondrial apoptosis, as evidenced by annexin V/propidium iodide staining and western blot analysis. Interestingly, ART-induced apoptosis diminished under iron-deficient conditions but intensified under iron-overload conditions. Taken together, these findings demonstrated the potential of ART in treating skin cancer through the induction of G0/G1 cell cycle arrest and iron-mediated mitochondrial apoptosis and supported further investigations in other test systems.     

Brucine,an effective natural compound derived from nux-vomica,induces G1 phase arrest and apoptosis in LoVo cells

Brucine is an alkaloid from nux vomica, has been shown various pharmacological actions. To study the possible anti-cancer mechanisms on LoVo cells, effects of Brucine on cell viability, cell cycle and apoptosis were investigated. The results showed that Brucine revealed strong growth inhibitory effect on LoVo cells, and caused LoVo cell shrinkage and membrane blobbing, induced cellular and DNA morphological changes. Cell cycle and apoptosis analysis documented that Brucine could change cell cycle and induce cell apoptosis. Brucine-mediated cell cycle arrest in G1 phase was associated with a marked increase of protein levels of CCND1 and decrease in CCNB1, cyclin E and CDC2. In addition, Brucine dose-dependently caused LoVo cells apoptosis evidenced by Annexin V/PI staining Brucine-induced apoptosis was mediated via up-regulation of Bax and down-regulation of Bcl-2. Furthermore, proteins Erk1/2, p38 and Akt phosphorylation were down regulated by Brucine in a dose-dependent manner. In summary, this paper indicates Brucine is effective against LoVo cells proliferation, and promotes LoVo cells death via apoptosis. These results reveal functional interplay among a series of pathway that are deregulated in cancer and suggest that their simultaneous targeting by Brucine could result in efficacious inhibition on cancer cells.     

B1, a novel topoisomerase II inhibitor, induces apoptosis and cell cycle G1 arrest in lung adenocarcinoma A549 cells

In our previous studies, we demonstrated that 2,6-bis-(2-chloroacetamido) anthraquinone (B1) showed a highly significant cytotoxic effect. However, its influence in the cell cycle and apoptotic induction effects has not been investigated yet. Here we report the antiproliferative effect of B1, for which IC50 values were 0.57 μmol/l for lung cancer A549 cells, 0.63 μmol/l for colon cancer HT-29 cells, and 0.53 μmol/l for breast cancer MCF-7 cells. DNA topoisomerase II (Topo II), an essential enzyme in DNA synthesis and meiotic division, is highly expressed in cancer cells. Some currently used clinical anticancer drugs (doxorubicin and mitoxantrone) targeting Topo II are very effective antineoplastic agents. B1, sharing the basic structure of known Topo II inhibitors, demonstrated a significant inhibitory effect on Topo II bioactivity. In A549 cells, B1 increased apoptotic cell population with induction of Fas, Bax, and cleaved poly(ADP-ribose) polymerase and by reduction of Bcl-2 expression. Moreover, cell cycle analysis indicated that B1 induced G1 phase arrest through modulation of G1 cell cycle regulatory proteins, such as the downregulation of cyclin D1 and upregulation of Cip/p21, Kip1/p27, and p53. Thus, our study suggests that B1, with the ability to inhibit Topo II activity and cause cell cycle G1 arrest and apoptosis, has potential as a novel anticancer agent.     

Ochratoxin A induces oxidative DNA damage and G1 phase arrest in human peripheral blood mononuclear cells in vitro

Ochratoxin A is one of the most abundant food-contaminating mycotoxins worldwide, and its immunosuppressive effects in human caused more and more concern in biomedical field. In the present study, the toxicity of OTA on human peripheral blood mononuclear cells (hPBMC) was explored by analyzing the involvement of oxidative pathway. It was found that OTA treatment led to the release of reactive oxygen species (ROS) and the increase of 8-hydroxydeoxyguanosine (8-OHdG), an important biomarker of oxidative DNA stress. Moreover, we found that OTA treatment induced DNA strand breaks in hPBMC as evidenced by DNA comet tails formation and increased γ-H2AX expression. In addition, OTA could induce cell cycle arrest at G1 phase by down-regulating the expression of CDK4 and cyclinD1 protein, as well as apoptosis in hPBMC in vitro. Pre-treatment of hPBMC with antioxidant, N-acetyl-L-cysteine (NAC), could reduce OTA-induced ROS release and DNA damage, thus confirming the involvement of oxidative DNA damage in the OTA genotoxicity in hPBMC. NAC pre-treatment could also significantly prevent OTA-induced down-regulation of CDK4 and cyclinD1 expression in hPBMC. All the results demonstrated the involvement of oxidative pathway in OTA mediated cytotoxicity in human immune cells, which including the ROS accumulation-oxidative DNA damage-G1 arrest and apoptosis. Our results provide new insights into the molecular mechanisms by which OTA might promote immunotoxicity.     

Molecular mechanisms of ZD1839-induced G1-cell cycle arrest and apoptosis in human lung adenocarcinoma A549 cells

Epithelial growth factor receptor (EGFR) has been proposed as a target for anticancer therapy. ZD1839 (Iressa) is a quinazoline derivative that selectively inhibits the EGFR tyrosine kinase activity and is under clinical use in cancer patients. However, the molecular mechanisms involved in ZD1839-mediated anticancer effects remain largely uncharacterized. In this study, exposure of human lung adenocarcinoma A549 cells to ZD1839 caused G1 arrest, and subsequently induced apoptosis. Moreover, ZD1839 increased the protein levels of p27(KIP1) and retinoblastoma-related Rb2/p130 while decreased the expression of cyclin-dependent kinase-2 (CDK2), CDK4, CDK6 and cyclin-D1, cyclin-D3. In vitro kinase assay showed that ZD1839 decreased these CDKs expression in A549 cells, leading to significantly reduce their kinase activities. In addition, ZD1839-induced death of A549 cells with characteristics of apoptosis including apoptotic morphological changes, DNA fragmentation and enhancement of TUNEL-positive cell. These events were accompanied by a marked increase of Fas protein expression, and activation of caspase-2, -3, -8. Co-treatment of cells with Fas antagonist antibody significantly blocked ZD1839-induced apoptosis. Caspase-8 and caspase-3 inhibitors, but not a caspase-9 inhibitor, were also capable of restoring cell viability. Our results indicate that downregulation of the expression and function of CDK2, CDK4, CDK6, cyclin-D1 and cyclin-D3, as well as upregulation of p27(KIP1) and pRb2/p130, are strong candidates for the cell cycle regulator that arrests ZD1839-treated A549 cells at G1 phase. Furthermore, upregulation of Fas appears to play a major role in the initiation of ZD1839-induced apoptosis, activation of caspase-8/caspase-3 cascade is involved in the execution phase of this death program.     

Aloe emodin induces G2/M cell cycle arrest and apoptosis via activation of caspase-6 in human colon cancer cells

Aloe emodin (AE), a natural anthraquinone, is reported to have antiproliferative activity in various cancer cell lines. In this study, we analyzed the molecular mechanisms involved in the growth-inhibitory activity of this hydroxyanthraquinone in colon cancer cell, WiDr. In our observation AE inhibited cell proliferation by arresting the cell cycle at the G2/M phase and inhibiting cyclin B1. AE appreciably induced cell death specifically through the induction of apoptosis and by activating caspases 9/6. Apoptotic execution was found to be solely dependent on caspase-6 rather than caspase-3 or caspase-7. This is the first study indicating that the AE induces apoptosis specifically through the activation of caspase-6.     

Effects of carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone on the growth inhibition in human pulmonary adenocarcinoma Calu-6 cells

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in eukaryotic cells. Here, we evaluated the in vitro effects of FCCP on the growth of Calu-6 lung cancer cells. FCCP inhibited the growth of Calu-6 cells with an IC₅₀ of approximately 6.64 ± 1.84 μM at 72 h, as shown by MTT. DNA flow cytometric analysis indicated that FCCP induced G1 phase arrest below 20 μM of FCCP. Treatment with FCCP decreased the level of CDKs and cyclines in relation to G1 phase. In addition, FCCP not only increased the p27 level but also enhanced its binding with CDK4, which was associated with hypophosphorylation of Rb protein. While transfection of p27 siRNA inhibited G1 phase arrest in FCCP-treated cells, it did not enhance Rb phosphorylation. FCCP also efficiently induced apoptosis. The apoptotic process was accompanied with an increase in sub-G1 cells, annexin V staining cells, mitochondria membrane potential (MMP) loss and cleavage of PARP protein. All of the caspase inhibitors (caspase-3, -8, -9 and pan-caspase inhibitor) markedly rescued the Calu-6 cells from FCCP-induced cell death. However, knock down of p27 protein intensified FCCP-induced cell death. Moreover, FCCP induced the depletion of GSH content in Calu-6 cells, which was prevented by all of the caspase inhibitors. In summary, our results demonstrated that FCCP inhibits the growth of Calu-6 cells in vitro. The growth inhibitory effect of FCCP might be mediated by cell cycle arrest and apoptosis via decrease of CDKs and caspase activation, respectively. These findings now provide a better elucidation of the mechanisms involved in FCCP-induced growth inhibition in lung cancer.     

Xanthatin induces G2/M cell cycle arrest and apoptosis in human gastric carcinoma MKN-45 cells

Xanthatin, a natural bioactive compound of sesquiterpene lactones, was isolated and purified from air-dried aerial part of Xanthium sibiricum Patrin ex Widder. In the present study, we demonstrated the significant antiproliferative and proapoptotic effects of xanthatin on human gastric carcinoma MKN-45 cells. MTS assay showed that xanthatin produced obvious cytotoxicity in MKN-45 cells with IC50 values of 18.6, 9.3, and 3.9 μM for 12, 24, and 48 h, respectively. Results of flow cytometry analysis indicated that the antiproliferative activity induced by xanthatin might be executed via G2/M cell cycle arrest and proapoptosis in MKN-45 cells. Western blot analysis elucidated that: a) xanthatin downregulated expression of Chk1 and Chk2 and phosphorylation of CDC2, which are known as key G2/M transition regulators; b) xanthatin increased p53 activation, decreased the bcl-2/bax ratio and the levels of downstream procaspase-9 and procaspase-3, which are key regulators in the intrinsic apoptosis pathway; c) xanthatin blocked phosphorylation of NF-κB (p65 subunit) and of IκBα, which might contribute to its proapoptotic effects on MKN-45 cells. In conclusion, our results suggest that xanthatin may have therapeutic potential against human gastric carcinoma.     

Quercetin simultaneously induces G0/G1‐phase arrest and caspase‐mediated crosstalk between apoptosis and autophagy in human leukemia HL‐60 cells

Quercetin is a plant‐derived bioflavonoid with high anticancer activity in various tumors. Herein, the molecular mechanisms by which quercetin exerts its anticancer effects against HL‐60 acute myeloid leukemia (AML) cells were investigated. Results showed that quercetin suppressed cell proliferation in the HL‐60 cell line in vitro and in vivo. Quercetin‐induced G ₀/G₁‐phase arrest occurred when expressions of cyclin‐dependent kinase (CDK)2/4 were inhibited and the CDK inhibitors, p16 and p21, were induced. Moreover, quercetin treatment not only activated proapoptotic signaling like poly (ADP ribose) polymerase (PARP)?1 cleavage and caspase activation but also triggered autophagy events as shown by the increased expression of light chain 3 (LC3)‐II, decreased expression of p62, and formation of acidic vesicular organelles. Interestingly, it was found that use of the autophagy inhibitor, 3‐methyladenine, significantly enhanced quercetin‐mediated apoptotic cell death as analyzed by MTS and DNA fragmentation assays. Moreover, pretreatment of HL‐60 cells with the pan‐caspase inhibitor, Z‐VAD‐fmk, dramatically reversed quercetin‐mediated apoptotic and autophagic cell death. Although apoptosis and autophagy are two independent cell death pathways, our findings indicated that quercetin can activate caspases to trigger these two pathways, and both pathways played contrary roles in quercetin‐mediated HL‐60 cell death. In conclusion, besides promoting apoptosis, quercetin also induced cytoprotective autophagy in HL‐60 cells, and inhibition of autophagy may be a novel strategy to enhance the anticancer activity of quercetin in AML.     

Osthole induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells

Context: Osthole [7-methoxy-8-(3-methyl-2-butenyl) coumarin] isolated from the fruit of Cnidium monnieri (L.) Cuss, one of the commonly used Chinese medicines listed in the Shennong’s Classic of Materia Medica in the Han Dynasty, had remarkable antiproliferative activity against human hepatocellular carcinoma HepG2 cells in culture.

Objectives: This study evaluated the effects of osthole on cell growth, nuclear morphology, cell cycle distribution, and expression of apoptosis-related proteins in HepG2 cells.

Materials and methods: Cytotoxic activity of osthole was determined by the MTT assay at various concentrations ranging from 0.004 to 1.0?µmol/ml in HepG2 cells. Cell morphology was assessed by Hoechst staining and fluorescence microscopy. Apoptosis and cell-cycle distribution was determined by annexin V staining and flow cytometry. Apoptotic protein levels were assessed by Western blot.

Results: Osthole exhibited significant inhibition of the survival of HepG2 cells and the half inhibitory concentration (IC₅₀) values were 0.186, 0.158 and 0.123?µmol/ml at 24, 48 and 72?h, respectively. Cells treated with osthole at concentrations of 0, 0.004, 0.02, 0.1 and 0.5?μmol/ml showed a statistically significant increase in the G2/M fraction accompanied by a decrease in the G0/G1 fraction. The increase of apoptosis induced by osthole was correlated with down-regulation expression of anti-apoptotic Bcl-2 protein and up-regulation expression of pro-apoptotic Bax and p53 proteins.

Conclusion: Osthole had significant growth inhibitory activity and the pro-apoptotic effect of osthole is mediated through the activation of caspases and mitochondria in HepG2 cells. Results suggest that osthole has promising therapeutic potential against hepatocellular carcinoma.     

5-Methoxyflavanone induces cell cycle arrest at the G2/M phase, apoptosis and autophagy in HCT116 human colon cancer cells

Natural flavonoids have diverse pharmacological activities, including anti-oxidative, anti-inflammatory, and anti-cancer activities. In this study, we investigated the molecular mechanism underlying the action of 5-methoxyflavanone (5-MF) which has a strong bioavailability and metabolic stability. Our results show that 5-MF inhibited the growth and clonogenicity of HCT116 human colon cancer cells, and that it activated DNA damage responses, as revealed by the accumulation of p53 and the phosphorylation of DNA damage-sensitive proteins, including ataxia-telangiectasia mutated (ATM) at Ser1981, checkpoint kinase 2 (Chk2) at Thr68, and histone H2AX at Ser139. 5-MF-induced DNA damage was confirmed in a comet tail assay. We also found that 5-MF increased the cleavage of caspase-2 and -7, leading to the induction of apoptosis. Pretreatment with the ATM inhibitor KU55933 enhanced 5-MF-induced γ-H2AX formation and caspase-7 cleavage. HCT116 cells lacking p53 (p53^−/−) or p21 (p21^−/−) exhibited increased sensitivity to 5-MF compared to wild-type cells. 5-MF further induced autophagy via an ERK signaling pathway. Blockage of autophagy with the MEK inhibitor U0126 potentiated 5-MF-induced γ-H2AX formation and caspase-2 activation. These results suggest that a caspase-2 cascade mediates 5-MF-induced anti-tumor activity, while an ATM/Chk2/p53/p21 checkpoint pathway and ERK-mediated autophagy act as a survival program to block caspase-2-mediated apoptosis induced by 5-MF.     

A novel adenosine analog, thio-Cl-IB-MECA, induces G0/G1 cell cycle arrest and apoptosis in human promyelocytic leukemia HL-60 cells

Human A3 adenosine receptor (A3AR) agonists have been shown to play important roles in several physiological and pathological processes, including growth inhibition of human cancer cells. On this line, we recently found that a novel adenosine analog, 2-chloro-N6-(3-iodobenzyl)-4'-thioadenosine-5'-N-methyluronamide (thio-Cl-IB-MECA) was a potent human A3AR agonist, and is superior to a known agonist Cl-IB-MECA [Jeong LS, Jin DZ, Kim HO, Shin DH, Moon HR, Gunaga P, et al. J Med Chem 2003;46:3775]. Here, we report that a novel A3AR agonist, thio-Cl-IB-MECA inhibited the growth of human promyelocytic leukemia HL-60 cells by arresting cell cycle and induction of apoptosis. Thio-Cl-IB-MECA induced the cell cycle arrest of G0/G1 in the early time and at lower concentration (up to 25 microM). At higher concentration (50 microM), the apoptotic cell deaths were manifested by observation of the increase of sub-G0 phase of cell cycle distribution, DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage. In addition, the down-regulation of checkpoint protein cyclin D1 and c-myc by thio-Cl-IB-MECA was well correlated with the arrest of cell cycle transition of G1 to S phase. Further study revealed that the growth inhibitory activity of thio-Cl-IB-MECA is also related with the modulation of Wnt signaling pathway. The levels of beta-catenin, phosphorylated forms of GSK-beta and Akt were down-regulated by the treatment of thio-Cl-IB-MECA (10 nM) in a time-dependent manner, providing one of plausible mechanistic evidence for the involvement of the Wnt signaling pathway in the HL-60 cell growth inhibitory effects by thio-Cl-IB-MECA. These results suggest that a novel A3AR agonist, thio-Cl-IB-MECA can down-regulate Wnt signaling, inhibit proliferation and induce apoptosis in HL-60 leukemia cells, and thus provide the possibility of this compound in the potential therapeutic value of the treatment of leukemia.     

Arecoline decreases interleukin-6 production and induces apoptosis and cell cycle arrest in human basal cell carcinoma cells

Arecoline, the most abundant areca alkaloid, has been reported to decrease interleukin-6 (IL-6) levels in epithelial cancer cells. Since IL-6 overexpression contributes to the tumorigenic potency of basal cell carcinoma (BCC), this study was designed to investigate whether arecoline altered IL-6 expression and its downstream regulation of apoptosis and the cell cycle in cultured BCC-1/KMC cells. BCC-1/KMC cells and a human keratinocyte cell line, HaCaT, were treated with arecoline at concentrations ranging from 10 to 100 μg/ml, then IL-6 production and expression of apoptosis- and cell cycle progress-related factors were examined. After 24 h exposure, arecoline inhibited BCC-1/KMC cell growth and decreased IL-6 production in terms of mRNA expression and protein secretion, but had no effect on HaCaT cells. Analysis of DNA fragmentation and chromatin condensation showed that arecoline induced apoptosis of BCC-1/KMC cells in a dose-dependent manner, activated caspase-3, and decreased expression of the anti-apoptotic protein Bcl-2. In addition, arecoline induced progressive and sustained accumulation of BCC-1/KMC cells in G2/M phase as a result of reducing checkpoint Cdc2 activity by decreasing Cdc25C phosphatase levels and increasing p53 levels. Furthermore, subcutaneous injection of arecoline led to decreased BCC-1/KMC tumor growth in BALB/c mice by inducing apoptosis. This study demonstrates that arecoline has potential for preventing BCC tumorigenesis by reducing levels of the tumor cell survival factor IL-6, increasing levels of the tumor suppressor factor p53, and eliciting cell cycle arrest, followed by apoptosis.     

Casuarinin from the bark of Terminalia arjuna induces apoptosis and cell cycle arrest in human breast adenocarcinoma MCF-7 cells

Casuarinin, a hydrolyzable tannin isolated from the bark of Terminalia arjuna L. (Combretaceae), was investigated for its antiproliferative activity in human breast adenocarcinoma MCF-7 cells. The results showed that casuarinin inhibited the proliferation of MCF-7 by blocking cell cycle progression in the G0/G1 phase and inducing apoptosis. An enzyme-linked immunosorbent assay showed that casuarinin increased the expression of p21/WAF1 concomitantly as the MCF-7 cells underwent G0/G1 arrest. An enhancement in Fas/APO-1 and its two forms of ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by casuarinin. Our study reports here for the first time that the induction of p21/WAF1 and the activity of Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of casuarinin in MCF-7 cells.