Benzobijuglone, a novel cytotoxic compound from Juglans mandshurica, induced apoptosis in HeLa cervical cancer cells

A new quinone compound, p-hydroxymethoxybenzobijuglone (HMBBJ), isolated from Juglans mandshurica by bioassay-guided fractionation, showed cytotoxic activity against HeLa cell line. Its chemical structure was determined by NMR and HREIMS spectra. In this paper, its ability to induce apoptosis in HeLa cells was studied for the first time. After treated with HMBBJ, the growth of HeLa cells was inhibited and cells displayed typical morphological apoptotic characteristics. Data from flow cytometry analysis showed that the HeLa cell cycle was arrested in the G2/M phase by HMBBJ, and the apoptotic rate of HeLa cells increased in a dose-dependent manner. Meanwhile, HMBBJ increased the expression of caspase-8, -3 and Bax, decreased the expression of Bcl-2, and lowered the ΔΨ_m. These findings reveal that HMBBJ could efficiently induce HeLa cells apoptosis through mitochondria dependent pathway and activation of the caspase cascade, and it may be a potential chemotherapeutic candidate for the treatment of cancer.     

hSHIP induces S-phase arrest and growth inhibition in cervical cancer HeLa cells

hSHIP a human SH2-containing inositol-5-phosphatase,acts as a negative regulator of proliferation and survival in hematopoietic cells.Therefore.hSHIP may play a crucial role in suppression of cervical cancer HeLa cells.In this study,pcDNA3.1-hSHIP-GFP plasmid was constructed and transfected into HeLa cells with Lipofectamine2000, stably transfected HeLa cells were established and their responses were investigated by Flow cytometry,MTT, tumorigenicity in nude mice,RT-PCR and ELISA assays.The results showed that the expression of hSHIP significantly induced S-phase arrest.cell growth inhibition.and down-regulation of Aktl/2 mRNA and p-Akt in HeLa cells.Our study supports an important role for hSHIP in suppression of cervical caucer HeLa cells.which may prove to be a novel therapeutic option for non-hematopoietic cancels.     

hSHIP induces S-phase arrest and growth inhibition in cervical cancer HeLa cells

hSHIP,a human SH2-containing inositol-5-phosphatase,acts as a negative regulator of proliferation and survival in hematopoietic cells.Therefore,hSHIP may play a crucial role in suppression of cervical cancer HeLa cells.In this study,pcDNA3.1-hSHIP-GFP plasmid was constructed and transfected into HeLa cells with Lipofectamine2000,stably transfected HeLa cells were established and their responses were investigated by Flow cytometry,MTT,tumorigenicity in nude mice,RT-PCR and ELISA assays.The results showed tha...     

Swertia chirayita suppresses the growth of non-small cell lung cancer A549 cells and concomitantly induces apoptosis via downregulation of JAK1/STAT3 pathway

Lung carcinoma is the leading cause of cancer-related mortalities worldwide, and present therapeutical interventions are not successful enough to treat this disease in many cases. Recent years have witnessed a surge in exploring natural compounds for their antiproliferative efficacy to expedite the characterization of novel anticancer chemotherapeutics. Swertia chirayita is a valued medicinal herb and possess intrinsic pharmaceutical potential. However, elucidation of its anticancer effects at molecular levels remains unclear and needs to be investigated. We assessed the anticancer and apoptotic efficacy of S. chirayita ethanolic extract (Sw-EtOH) on non-small cell lung cancer (NSCLC) A549 cells during this exploratory study. The results elucidated that S. chirayita extract induced toxic effects within lung cancer cells by ~1 fold during cytotoxicity and LDH release assay at a 400 μg/ml concentration. Sw-EtOH extract elevates the level of ROS, resulting in the disruption of Δψm and release of cytosolic cytochrome c by 3.15 fold. Activation of caspases-3, -8 & -9 also escalated by ~1 fold, which further catalyze the augmentation of PARP cleavage (~3 folds), resulting in a four-fold increase in Sw-EtOH induced apoptosis. The gene expression analysis further demonstrated that Sw-EtOH extracts inhibited JAK1/STAT3 signaling pathway by down-regulating the levels of JAK1 and STAT3 to nearly half a fold. Treatment of Sw-EtOH modulates the expression level of various STAT3 associated proteins, including Bcl-XL, Bcl-2, Mcl-1, Bax, p53, Fas, Fas-L, cyclinD1, c-myc, IL-6, p21 and p27 in NSCLC cells. Thus, our study provided a strong impetus that Sw-EtOH holds the translational potential of being further evaluated as efficient cancer therapeutics and a preventive agent for the management of NSCLC.     

Morusin induces apoptosis and suppresses NF-kappaB activity in human colorectal cancer HT-29 cells

Morusin is a pure compound isolated from root bark of Morusaustralis (Moraceae). In this study, we demonstrated that morusin significantly inhibited the growth and clonogenicity of human colorectal cancer HT-29 cells. Apoptosis induced by morusin was characterized by accumulation of cells at the sub-G₁ phase, fragmentation of DNA, and condensation of chromatin. Morusin also inhibited the phosphorylation of IKK-α, IKK-β and IκB-α, increased expression of IκB-α, and suppressed nuclear translocation of NF-κB and its DNA binding activity. Dephosphorylation of NF-κB upstream regulators PI3K, Akt and PDK1 was also displayed. In addition, activation of caspase-8, change of mitochondrial membrane potential, release of cytochrome c and Smac/DIABLO, and activation of caspase-9 and -3 were observed at the early time point. Downregulation in the expression of Ku70 and XIAP was exhibited afterward. Caspase-8 or wide-ranging caspase inhibitor suppressed morusin-induced apoptosis. Therefore, the antitumor mechanism of morusin in HT-29 cells may be via activation of caspases and inhibition of NF-κB.     

Ginsenoside Rd from Panax notoginseng is cytotoxic towards HeLa cancer cells and induces apoptosis

The saponin ginsenoside Rd (1), isolated from Panax notoginseng, is used for the treatment of cardiovascular diseases, inflammation, different body pains, trauma, and internal and external bleeding due to injury. In this study, we report that 1 inhibits the cell growth of human cervical cancer (HeLa) cells in a concentration- and time-dependent manner, with an IC(50) value of 150.5+/-0.8 mcirog/ml after 48 h of incubation. The drug-treated cells displayed features of apoptosis, including typical morphological characteristics and formation of DNA ladders, as evident from agarose-gel electrophoresis. Flow-cytometric analysis showed that the cell-cycle distribution of HeLa cells exposed to 1 is characterized by a decrease of the G(0)/G(1)-phase and an increase of the S-phase cells, respectively, in a dose-dependent manner. The apoptotic rate of HeLa cells treated for 48 h with 210 microg/ml of 1 was 35.8%. Further, 1 was found to increase the expression of Bax and to decrease the expression of Bcl-2 proteins, respectively, and to lower the mitochondrial transmembrane potential of HeLa cells. The caspase-3 inhibitor DEVD-CHO (at 2 microM) increased the viability of HeLa cells treated with 1. Taken together, our study suggests that ginsenoside Rd (1) significantly inhibits HeLa cell proliferation, and induces cell apoptosis through down-regulating Bcl-2 expression, up-regulating Bax expression, lowering the mitochondrial transmembrane potential, and activating the caspase-3 pathway. Thus, 1 could serve as a lead to develop novel chemotherapeutic or chemopreventive agents against human cervical cancer.     

Apoptosis effects of Xrel3 c-Rel/Nuclear Factor-kappa B homolog in human cervical cancer cells

Cervical cancer is considered a common yet preventable cause of death in women. In this report, we studied the role of the NF-kappaB gene family in HeLa human cervical cancer cells, using the Xrel3 c-Rel homologue of Xenopus laevis. The expression of Xrel3/c-Rel slowed cell growth 6-fold, consistent with an upregulated expression of the cell cycle inhibitor p21. The activated PARP apoptosis effector was significantly increased (P<0.01). Based on cell viability assays Xrel3 provided an anti-apoptotic effect in 1 microM cisplatin, and this was associated with significantly lower levels of the apoptotic proteins Bax and MDM-2 (P<0.05). Furthermore, there was a 3-fold drop in the level of the tumor suppressor protein p53. In 5 microM cisplatin, expression of HeLa Xrel3 enhanced apoptosis by significantly increasing the expression of the apoptotic proteins Bax and MDM-2 (P<0.05). However, the tumor suppressor protein p53 showed a significant decrease (P<0.05) relative to the control. Thus, c-Rel/NF-kappaB may potentially be of clinical significance, especially in tumors exhibiting resistance to high-level chemotherapy.     

Hydrogen peroxide induces apoptosis in HeLa cells through mitochondrial pathway

Cervical cancer is the most common cancer amongst females in India and is associated with high risk HPVs, reactive oxygen species (ROS), and excessive inflammation in most cases. ROS in turn affects the expression of pro- and anti-apoptotic proteins. The objective of the present study was to elucidate the effect of hydrogen peroxide (H(2)O(2)) on apoptotic signaling molecules in vitro. HeLa cell line expresses the Human papilloma virus - 18, E6 oncoprotein which causes the ubiquitin mediated degradation of p53 protein and is thus p53 deficient. p53 is known to act as a cellular stress sensor and triggers apoptosis. p73, a member of the p53 family also induces apoptosis in response to DNA damaging agents but unlike p53, it is infrequently mutated in human tumors. We demonstrate here, that in HeLa cells, apoptosis is triggered by H(2)O(2) via the mitochondrial pathway involving upregulation of p73, and its downstream target Bax. This was accompanied by upregulation of ERK, JNK, c-Myc, Hsp-70 and down regulation of anti-apoptotic Bcl-XL, release of cytochrome c from mitochondria and activation of caspases-9 and -3.     

Inhibition of JAK2/STAT3 signalling induces colorectal cancer cell apoptosis via mitochondrial pathway

Abnormalities in the JAK2/STAT3 pathway are involved in the pathogenesis of colorectal cancer (CRC), including apoptosis. However, the exact mechanism by which dysregulated JAK2/STAT3 signalling contributes to the apoptosis has not been clarified. To investigate the role of both JAK2 and STAT3 in the mechanism underlying CRC apoptosis, we inhibited JAK2 with AG490 and depleted STAT3 with a small interfering RNA. Our data showed that inhibition of JAK2/STAT3 signalling induced CRC cellular apoptosis via modulating the Bcl-2 gene family, promoting the loss of mitochondrial transmembrane potential (Δψm) and the increase of reactive oxygen species. In addition, our results demonstrated that the translocation of cytochrome c (Cyt c), caspase activation and cleavage of poly (ADP-ribose) polymerase (PARP) were present in apoptotic CRC cells after down-regulation of JAK2/STAT3 signalling. Moreover, inhibition of JAK2/STAT3 signalling suppressed CRC xenograft tumour growth. We found that JAK2/STAT3 target genes were decreased; meanwhile caspase cascade was activated in xenograft tumours. Our findings illustrated the biological significance of JAK2/STAT3 signalling in CRC apoptosis, and provided novel evidence that inhibition of JAK2/STAT3 induced apoptosis via the mitochondrial apoptotic pathway. Therefore, JAK2/STAT3 signalling may be a potential target for therapy of CRC.     

低强度超声对HeLa肿瘤细胞凋亡的诱导作用及其机制

目的探讨低强度超声在诱导HeLa肿瘤细胞凋亡中的作用及其机制。 方法将体外培养的HeLa肿瘤细胞根据超声干预强度分为阴性对照组（切断电源后给予假的超声干预）和0.5、1.3、2.0 W/cm²超声干预组,分别通过MTT实验测定细胞活力、存活率,HE染色检测细胞形态,细胞凋亡实验检测细胞凋亡率,流式细胞术测定ROS含量及Western blot实验检测caspase-12、survivin和内参蛋白β-actin的表达情况。多组间比较采用单因素方差分析,各个实验组与对照组比较采用Dunnet-t检验。 结果与阴性对照组比较,0.5、1.3、2.0 W/ cm²超声干预组的HeLa肿瘤细胞活力[（99.23±1.56）﹪比（80.52±1.72）﹪、（54.31±1.69）﹪（27.75±1.26）﹪]、细胞存活率[（99.91±1.51）﹪比（76.69±1.92）﹪、（52.57±1.63）﹪、（29.81±1.22）﹪]、survivin蛋白表达（51.19±0.21比43.46±0.34、25.28±0.29、18.32±0.3）均降低,ROS含量（11.21±0.45比24.34±1.23、38.26±2.47、52.18±1.56）,细胞凋亡率[（4.23±1.21）﹪比（24.16±1.91）﹪、（48.34±1.66）﹪、（70.27±0.98）﹪]、caspase-12蛋白表达（13.05±0.21比20.23±0.19、33.17±0.32、41.52±0.21）均升高,差异具有统计学意义（P均< 0.05）。 结论低强度超声可能通过诱导HeLa肿瘤细胞中caspase-12蛋白表达增加和survivin蛋白表达的减少而使HeLa肿瘤细胞发生凋亡。     

An ROS generator, antimycin A, inhibits the growth of HeLa cells via apoptosis

Antimycin A (AMA), an inhibitor of electron transport in mitochondria, has been used as a reactive oxygen species (ROS) generator in biological systems. Here, we investigated the in vitro effect of AMA on apoptosis in HeLa cells. AMA inhibited the growth of HeLa cells with an IC(50) of about 50 microM. AMA efficiently induced apoptosis, as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay, and DAPI staining. This apoptotic process was accompanied by the loss of mitochondrial membrane potential (DeltaPsi(m)), Bcl-2 down-regulation, Bax up-regulation, and PARP degradation. All caspase inhibitors used in this experiment, especially pan-caspase inhibitor (Z-VAD), could rescue some HeLa cells from AMA-induced cell death. When we examined the changes of the ROS, H(2)O(2) or O(2) (.-), in AMA-treated cells, H(2)O(2) and O(2) (.-) were markedly increased. In addition, we detected the depletion of GSH content in AMA-treated cells. Pan-caspase inhibitor showing the efficient anti-apoptotic effect significantly reduced GSH depletion by AMA. Superoxide dismutase (SOD) and catalase did not reduce intracellular ROS, but these could strongly rescue the cells from apoptosis. However, these anti-apoptotic effects were not accompanied by the recovery of GSH depletion. Interestingly, catalase significantly decreased the CMF negative (GSH depletion) and propidium iodide (PI) positive cells, indicating that catalase strongly maintained the integrity of the cell membrane in CMF negative cells. Taken together, these results demonstrate that AMA potently generates ROS, induces the depletion of GSH content in HeLa cells, and strongly inhibits the growth of HeLa cells throughout apoptosis.     

Dual apoptotic effect of Xrel3 c-Rel/NF-kappaB homolog in human cervical cancer cells

Cervical cancer is one of the most common cancers affecting a woman's reproductive organs. Despite its frequency and recurrence, the death rate has been declining over the past 40 years, due to early detection and treatment. In a previous report [Shehata Marlene, Shehata Marian, Shehata Fady, Pater Alan. Apoptosis effects of Xrel3 c-Rel/Nuclear factor-kappa B homolog in human cervical cancer cells. Cell Biology International, in press], we studied the role of the NF-kappaB gene family in HeLa human cervical cancer cells, using the Xrel3 c-Rel homologue of Xenopus laevis. These results showed that the expression of Xrel3/c-Rel slowed cell growth, consistent with an upregulated expression of the cell cycle inhibitor p21 and the activated poly(ADP-ribose) polymerase (PARP) apoptosis effector. However, in this report, we examined more apoptotic and anti-apoptotic factors acting upstream and downstream in apoptosis pathways after cisplatin treatment of HeLa cervical cancer cells. After 1 microM cisplatin treatment, Xrel3 had an anti-apoptotic effect, based on significantly lower levels of apoptotic proteins, including caspase-8, caspase-3 and p21. Anti-apoptotic BAG-1 isoforms were upregulated. After 5 microM cisplatin treatment, expression of HeLa Xrel3 had an apoptotic effect, based on significantly increased expression of the cell cycle inhibitor p21 and apoptotic proteins, including cleaved PARP, caspase-8, and caspase-3. However, anti-apoptotic Bcl-2 and Bcl-X(L) were elevated and the cell cycle regulator cyclin D1 was slightly upregulated with both 1 and 5 microM cisplatin treatment. The HPV E6 oncoprotein showed no significant changes. These results support previous conclusions on the potential anti-apoptotic effects of c-Rel/NF-kappaB in mild stress environments, as opposed to the apoptotic effects associated with high stress conditions [Lake BB, Ford R, Kao KR. Xrel3 is required for head development in Xenopus laevis. Development 2001; 128(2), 263-73.]. Thus, c-Rel/NF-kappaB may potentially be of clinical significance in chemotherapy.     

Punicalagin promotes the apoptosis in human cervical cancer (ME-180) cells through mitochondrial pathway and by inhibiting the NF-kB signaling pathway

Increasing attention of plant derived therapeutic agents against cancer, investigating the anti-proliferative efficiency of plant derived chemicals have achieved increasing momentum for the design of anticancer drug. Punicalagin, dietary phytochemical altered the various cell signal transduction pathways associated with cell apoptosis and proliferation. This investigation was intended to examine the efficiency of punicalagin lying on cell viability so as to examine the molecular based punicalagin mechanism stimulated apoptosis via exploring the expression of Bcl-2 family proteins, and caspases also the cell cycle regulatory proteins p53 and NF-κB signaling in human cervical cancer cells. We also analyzed the morphological characteristic changes through mitochondrial membrane depolarization, reactive oxygen species (ROS) generation, TUNEL assay, AO/EtBr analysis in cervical cancer cells. Our findings demonstrated that punicalagin repressed the viability of cervical cancer cells in a dosereliant mode via stimulating mitochondrial mediated apoptosis. Moreover, our this study demonstrated that punicalagin blocked cervical cancer cell proliferation and stimulated cell apoptosis by suppressing NF-kappa B activity. Hence our study suggested that punicalagin exhibits opposing actions on NF-kappa B signaling networks to block cancer cell progression acts as a classical candidate for anticancer drug designing.     

Ge-Jee-Bok-Ryung-Hwan induces apoptosis in human cervical carcinoma HeLa cells--an endoplasmic reticulum stress pathway--

Ge-Jee-Bok-Ryung-Hwan (GJBRH), a commonly used herb formulation in Korea, Japan and China, caused a decrease of viability in HeLa human cervical carcinoma cells. The treatment of GJBRH resulted in genomic DNA fragmentation as well as the increase of Sub-G1 portion in cell cycle analysis. In this study, GFP-Bax over-expression system showed that Bax, pro-apoptotic Bcl-2 family protein, was translocated to mitochondria by the presence of GJBRH. The treatment of BAPTA-AM, permeable endogenous calcium chelator, inhibited GJBRH-induced caspase-3 and -9 activations, the release of cytochrome c and Smac/DIABLO into cytoplasm and the resultant cell death in HeLa human cervical carcinoma cells. The treatment of BAPTA-AM increased the expression of XIAP, which mediates binding to and inhibiting caspases and showed protective effect, in GJBRH-treated cells. GJBRH induced the expression of Glucose Response Protein 78 (GRP 78), a positive ER stress marker protein. However, BAPTA-AM did not interfere with the ER-stress response pathway that triggers the expression of GRP 78. This study showed that GJBRH induces cell death, which occurs downstream of or parallel to this point in the ER-stress pathway linked to apoptosis. In conclusion, GJBRH induces apoptosis in HeLa cells via ER stress-pathway associated mitochondria-dependent apoptosis mechansim.     

CMTM5-v1 induces apoptosis in cervical carcinoma cells

CMTM5 (CKLF-like MARVEL transmembrane domain-containing member 5) exhibits tumor inhibition activity with frequent epigenetic inactivation in various tumor cell lines including cervical carcinoma (CC) cells. In this paper, we examined the function of CMTM5-v1 (the primary RNA splicing form) in both HeLa and SiHa cells. Overexpression of CMTM5-v1 in both cells can induce apoptosis, but the effects are more obvious in SiHa than that in HeLa. In SiHa cells, restoration of CMTM5-v1 caused disruption of mitochondrial transmembrane potential, release of cytochrome c, activation of caspase3 and cleavage of PARP. General caspase inhibitor almost prevented apoptosis of SiHa cells, suggesting that CMTM5-v1 induces apoptosis mainly through caspase-dependent pathway. These findings verify that CMTM5-v1 inhibits the growth of CC cell lines via inducing apoptosis.     

Hispolon induces apoptosis in human gastric cancer cells through a ROS-mediated mitochondrial pathway

Severe side effects and complications such as gastrointestinal and hematological toxicities because of current anticancer drugs are major problems in the clinical management of gastric cancer, which highlights the urgent need for novel effective and less toxic therapeutic approaches. Hispolon, an active polyphenol compound, is known to possess potent antineoplastic and antiviral properties. In this study, we investigated the efficacy of hispolon in human gastric cancer cells and explored the cell death mechanism. Hispolon induced ROS-mediated apoptosis in gastric cancer cells and was more toxic toward gastric cancer cells than toward normal gastric cells, suggesting greater susceptibility of the malignant cells. The mechanism of hispolon-induced apoptosis was that hispolon abrogated the glutathione antioxidant system and caused massive ROS accumulation in gastric cancer cells. Excessive ROS caused oxidative damage to the mitochondrial membranes and impaired the membrane integrity, leading to cytochrome c release, caspase activation, and apoptosis. Furthermore, hispolon potentiated the cytotoxicity of chemotherapeutic agents used in the clinical management of gastric cancer. These results suggest that hispolon could be useful for the treatment of gastric cancer either as a single agent or in combination with other anticancer agents.     

Betulinic acid induces apoptosis and inhibits metastasis of human colorectal cancer cells in vitro and in vivo

Betulinic acid (BA) is a pentacyclic triterpenoids extracted from birch with a wide range of biological properties. Recent studies have shown that BA has significant cytotoxicity to various types of human cancer cells, and shows potential in cancer treatment. However, the efficacy of BA on human colorectal cancer tumor cells is still unclear. The purpose of our study was to evaluate the anti-cancer activity of BA in human colorectal cancer cells in vitro and in vivo to investigate the possible mechanism. In this experiment, we found that BA inhibited colorectal cancer cell lines in vitro with a time-dependent and dose-dependent manner. Moreover, BA could induce cell apoptosis by upregulating expression of Bax and cleaved caspase-3 and downregulating protein of Bcl-2. BA could increase the production of reactive oxygen species and reduce mitochondrial membrane potential of cancer cell, suggesting that BA induced cancer cells apoptosis by mitochondrial mediated pathways. Furthermore, BA significantly inhibited the migration and invasion of colorectal cancer cells, reduced the expression of matrix metalloproteinase (MMPs) and increased the expression of MMPs inhibitor (TIMP-2). In addition, the growth of tumor was significantly suppressed by intraperitoneal administration of 20 mg/kg/day of BA in a xenograft tumor mouse model of HCT-116. Histopathological and immunohistochemical analysis showed that MMP-2+ cells and Ki-67+ cells were reduced and cleaved caspase-3+ cells were increased in tumor tissues of mice after BA administration. The results showed that BA not only promoted the apoptosis of colorectal cancer cells, but also inhibited the metastasis of cancer cells. Our results suggest that BA can be a potential natural drug to inhibit the growth and metastasis of colorectal cancer.     

Vitamin E succinate inhibits survivin and induces apoptosis in pancreatic cancer cells

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States. Identifying novel chemotherapeutic and chemopreventive approaches is critical in the prevention and treatment of cancers such as pancreatic cancer. Vitamin E succinate (VES) is a redox-silent analog of the fat-soluble vitamin alpha-tocopherol. In the present study, we explored the antiproliferative action of VES and its effects on inhibitor of apoptosis proteins in pancreatic cancer cells. We show that VES inhibits cell proliferation and induces apoptosis in pancreatic cancer cells. Further, we demonstrate that VES downregulates the expression of survivin and X-linked inhibitor of apoptosis proteins. The apoptosis induced by VES was augmented by siRNA-mediated inhibition of survivin in PANC-1 cells. In summary, our results suggest that VES targets survivin signaling and induces apoptosis in pancreatic cancer cells.