Gomisin A ameliorates metastatic melanoma by inhibiting AMPK and ERK/JNK-mediated cell survival and metastatic phenotypes

BackgroundGomisin A (G.A), a lignan compound extracted from the fruits of Schisandra chinensis, is known to exert anti-tumor effects on hepatocarcinoma and colorectal cancer cells. Suppression of proliferation and metastatic abilities of cancer cells are some effective cancer treatment methods.PurposeThe objective of this study is to investigate the effects of G.A on metastatic melanoma, and the mechanism by which it affects metastatic melanoma.Study designThe anti-proliferative and anti-metastatic effects of G.A were observed in in vitro and in vivo.MethodsWST assay and flow cytometry were conducted to investigate the effect of G.A on proliferation, cell cycle arrest, and apoptosis in metastatic melanoma cell lines. Migration and invasion abilities of G.A-treated melanoma cells were observed by wound healing and invasion assays.ResultsG.A (25–100 μM) decreased the viability of melanoma cells by inducing cell cycle arrest and apoptosis. These anti-proliferative effects of G.A were found to be mediated by AMPK, ERK, and JNK activation. G.A (5–20 μM) decreased the migration and invasion of melanoma cells by suppressing epithelial-mesenchymal transition (EMT). Consequently, G.A (2–50 mg/kg) inhibited lung metastasis by suppressing EMT and inducing cell cycle arrest and apoptosis in melanoma cells.ConclusionThese results conclude that G.A has the potential to reduce metastatic melanoma through its anti-proliferative and anti-metastatic effects.     

Inhibition of PI3K/AKT signaling via ROS regulation is involved in Rhein-induced apoptosis and enhancement of oxaliplatin sensitivity in pancreatic cancer cells

Several natural products have been demonstrated to both enhance the anti-tumor efficacy and alleviate the side effects of conventional chemotherapy drugs. Rhein, a main constituent of the Chinese herb rhubarb, has been shown to induce apoptosis in various cancer types. However, the exact pharmacological mechanisms controlling the influence of Rhein on chemotherapy drug effects in pancreatic cancer (PC) remain largely undefined. In this study, we found that Rhein inhibited the growth and proliferation of PC cells through G1 phase cell cycle arrest. Moreover, Rhein induced caspase-dependent mitochondrial apoptosis of PC cells through inactivation of the PI3K/AKT pathway. Combination treatment of Rhein and oxaliplatin synergistically enhanced apoptosis of PC cells through increased generation of intracellular reactive oxygen species (ROS) and inactivation of the PI3K/AKT pathway. Pre-treatment with the ROS scavenger N-acetyl-L-cysteine attenuated the combined treatment-induced apoptosis and restored the level of phosphorylated AKT, indicating that ROS is an upstream regulator of the PI3K/AKT pathway. The combination therapy also exhibited stronger anti-tumor effects compared with single drug treatments in vivo. Taken together, these data demonstrate that Rhein can induce apoptosis and enhance the oxaliplatin sensitivity of PC cells, suggesting that Rhein may be an effective strategy to overcome drug resistance in the chemotherapeutic treatment of PC.     

Stimulation of autophagic activity in human glioma cells by anti-proliferative ardipusilloside I isolated from Ardisia pusilla

Aims

Ardipusilloside I (ADS-I), a triterpenoid saponin isolated from Ardisia pusilla A.DC (Myrsinaceae), has been recently tested for cancer treatment including brain cancer. However, the mechanism of its action remains elusive. The present study was to investigate the role of autophagy activation in the anti-tumor activities of ADS-I in human glioma cells.

Main methods

The tetrazolium dye (MTT) colorimetric assay was used for the measurement of cell proliferation in cultured glioma cells, transmission electron microscopy (TEM) for the examination of autophagic activity, flow cytometric analysis for the determination of cell cycle and apoptotic cells, and immunocytochemistry and Western blot for protein expression of microtubule-associated protein light-chain 3 (LC3) and Beclin 1.

Key findings

ADS-I significantly inhibited the proliferation of both U373 and T98G glioma cells in cultures in a dose-dependent manner. The cytotoxic activity of ADS-I against glioma cell growth was associated not only with the induction of cell cycle arrest at G₂/M phase and cell apoptosis in flow cytometric analysis, but also with the activation of autophagy, indicated by the formation of autophagosomes and up-regulated expression of both autophagic protein Beclin 1 and LC3 in glioma cells. Additionally, the treatment with chloroquine, an autophagy inhibitor, reduced ADS-1-mediated cell death.

Significance

These data suggest that the anti-proliferative activity of ADS-I in human glioma cells is associated with the activation of autophagy in addition to cell cycle arrest and apoptosis, and the antagonistic effect of chloroquine suggests an important role of autophagy in ADS-I-mediated cell death against tumor growth.     

Cardamonin induces G2/M arrest and apoptosis via activation of the JNK–FOXO3a pathway in breast cancer cells

Cardamonin (CD), a naturally occurring chalcone isolated from large black cardamom, was previously reported to suppress the proliferation of breast cancer cells. However, its precise molecular anti‐tumor mechanisms have not been well elucidated. In this study, we found that CD markedly inhibited the proliferation of MDA‐MB 231 and MCF‐7 breast cancer cells through the induction of G2/M arrest and apoptosis. Reactive oxygen species (ROS) plays a pivotal role in the inhibition of CD‐induced cell proliferation. Treatment with N‐acetyl‐cysteine (NAC), an ROS scavenger, blocked CD‐induced G2/M arrest and apoptosis in this study. Quenching of ROS by overexpression of catalase also blocked CD‐induced cell cycle arrest and apoptosis. We showed that CD enhanced the expression and nuclear translocation of Forkhead box O3 (FOXO3a) via upstream c‐Jun N‐terminal kinase, inducing the expression of FOXO3a and its target genes, including p21, p27, and Bim. This process led to the reduction of cyclin D1 and enhancement of activated caspase‐3 expression. The addition of NAC markedly reversed these effects, knockdown of FOXO3a using small interfering RNA also decreased CD‐induced G2/M arrest and apoptosis. In vivo, CD efficiently suppressed the growth of MDA‐MB 231 breast cancer xenograft tumors. Taken together, our data provide a molecular mechanistic rationale for CD‐induced cell cycle arrest and apoptosis in breast cancer cells.     

Berberine, a genotoxic alkaloid, induces ATM-Chk1 mediated G2 arrest in prostate cancer cells

Berberine has been shown to possess anti-tumor activity against a wide spectrum of cancer cells. It inhibits cancer cell proliferation by inducing cell cycle arrest, at G1 and/or G2/M, and apoptosis. While it has been documented that berberine induces G1 arrest by activating the p53-p21 cascade, it remains unclear what mechanism underlies the berberine-induced G2/M arrest, which is p53-independent. In this study, we tested the anti-proliferative effect of berberine on murine prostate cancer cell line RM-1 and characterized the underlying mechanisms. Berberine dose-dependently induced DNA double-strand breaks and apoptosis. At low concentrations, berberine was observed to induce G1 arrest, concomitant with the activation of p53-p21 cascade. Upon exposure to berberine at a higher concentration (50μM) for 24h, cells exhibited G2/M arrest. Pharmacological inhibition of ATM by KU55933, or Chk1 by UCN-01, could efficiently abrogate the G2/M arrest in berberine-treated cells. Downregulation of Chk1 by RNA interference also abolished the G2/M arrest caused by berberine, confirming the role of Chk1 in the pathway leading to G2/M arrest. Abrogation of G2/M arrest by ATM inhibition forced more cells to undergo apoptosis in response to berberine treatment. Chk1 inhibition by UCN-01, on the other hand, rendered cells more sensitive to berberine only when p53 was inhibited. Our results suggest that combined administration of berberine and caffeine, or other ATM inhibitor, may accelerate the killing of cancer cells.     

Rhopaloic acid A induces apoptosis,autophagy and MAPK activation through ROS-mediated signaling in bladder cancer

BackgroundBladder cancer (BC) is a very common type of malignant cancer in men and new therapeutic strategies are urgently needed to reduce mortality. Several studies have demonstrated that Rhopaloic acid A (RA), a compound isolated from marine sponges, fights cancer but its potential anti-tumor effect on BC is still unknown.PurposeThe present study was aimed to explore the potential anti-tumor effects of RA against human BC cells and the underlying molecular mechanism.MethodsCell cytotoxicity was determined using the MTT and colony formation assays. Cell cycle distribution, apoptosis induction and generation of mitochondrial reactive oxygen species (ROS) were analyzed by flow cytometry. Mitochondrial membrane potential, acridine orange staining and intracellular ROS levels were observed using fluorescence microscopy. Levels of various signaling proteins were assessed using Western blotting. Furthermore, a zebrafish BC xenotransplantation model was used to confirm the anti-tumor effect of RA in vivo.ResultsTreatment with RA significantly suppressed the proliferation of BC cells that resulted from G2/M cycle arrest. Additionally, RA induced mitochondrial-mediated apoptosis and autophagy in BC cells. The death of BC cells induced by RA was rescued by treatment with inhibitors of apoptosis (Z-VAD-FMA) or autophagy (3-MA). RA activated the MAPK pathway and increased the production of cellular and mitochondrial ROS. Treatment with the ROS scavenger N-acetyl cysteine, effectively reversed the induction of apoptosis, autophagy, JNK activation and DNA damage elicited by RA. Finally, RA significantly inhibited tumor growth in a zebrafish BC xenotransplantation model.ConclusionTaken together, our findings indicate that RA induces apoptosis and autophagy and activates the MAPK pathway through ROS-mediated signaling in human BC cells. This RA-induced pathway offers insights into the molecular mechanism of its antitumor effect and shows that RA is a promising candidate for the treatment of BC.     

Anti-tumor activity of the X-linked inhibitor of apoptosis (XIAP) inhibitor embelin in gastric cancer cells

This study investigated the anticancer effects of embelin in human gastric cancer cells and the underlying molecular mechanisms. Gastric cancer cells were treated with embelin and 5-FU for methyl-thiazolyl-tetrazolium bromide cell viability assay and flow cytometric detection of cell viability and apoptosis. Protein pathway array (PPA) and Western blot were used to investigate differentially expressed proteins in embelin-treated gastric cancer cells. Embelin reduced gastric cancer cell viability, induced apoptosis, and enhanced 5-FU antitumor activity in gastric cancer cells. Mechanistically, embelin induced cell cycle arrest at the S and G2/M phases. Molecularly, embelin downregulated expression of X-linked inhibitor of apoptosis and cell cycle-regulatory proteins, such as CDK1, CDC25B, CDC25C, cyclinB1, and CDK2. PPA analysis showed that embelin modulated several pathways that are associated with cell growth and apoptosis, such as PI3K/AKT, JAK/STAT, p38 MAPK, and p53. The data from the current study implied that reduction of gastric cancer cell viability after treatment with embelin was through cell cycle arrest at the S and G2/M phases and apoptosis.     

Licarin A induces cell death by activation of autophagy and apoptosis in non-small cell lung cancer cells

Lung cancer has a relatively poor prognosis with a low survival rate and drugs that target other cell death mechanism like autophagy may help improving current therapeutic strategy. This study investigated the anti-proliferative effect of Licarin A (LCA) from Myristica fragrans in non-small cell lung cancer cell lines—A549, NCI-H23, NCI-H520 and NCI-H460. LCA inhibited proliferation of all the four cell lines in a dose and time dependent manner with minimum IC50 of 20.03?±?3.12, 22.19?±?1.37 µM in NCI-H23 and A549 cells respectively. Hence NCI-H23 and A549 cells were used to assess the ability LCA to induce autophagy and apoptosis. LCA treatment caused G1 arrest, increase in Beclin 1, LC3II levels and degradation of p62 indicating activation of autophagy in both NCI-H23 and A549 cells. In addition, LCA mediated apoptotic cell death was confirmed by MMP loss, increased ROS, cleaved PARP and decreased pro-caspase3. To understand the role of LCA induced autophagy and its association with apoptosis, cells were analysed following treatment with a late autophagy inhibitor-chloroquine and also after Beclin 1 siRNA transfection. Data indicated that inhibition of autophagy resulted in reduced anti-proliferative as well as pro-apoptotic ability of LCA. These findings confirmed that LCA brought about autophagy dependent apoptosis in non-small cell lung cancer cells and hence it may serve as a potential drug candidate for non-small cell lung cancer therapy.     

Triazole analog 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol induces reactive oxygen species and autophagy-dependent apoptosis in both in vitro and in vivo breast cancer models

Autophagy is considered as an important cell death mechanism that closely interacts with other common cell death programs like apoptosis. Critical role of autophagy in cell death makes it a promising, yet challenging therapeutic target for cancer. We identified a series of 1,2,3-triazole analogs having significant breast cancer inhibition property. Therefore, we attempted to study whether autophagy and apoptosis were involved in the process of cancer cell inhibition. The lead molecule, 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol (T-12) induced significant cell cycle arrest, mitochondrial membrane depolarization, apoptosis and autophagy in MCF-7 and MDA-MB-231 cells. T-12 increased reactive oxygen species and its inhibition by N-acetyl-l-cysteine protected breast cancer cells from autophagy and apoptosis. Autophagy inhibitor, 3-methyladenine abolished T-12 induced apoptosis, mitochondrial membrane depolarization and reactive oxygen species generation. This suggested that T-12 induced autophagy facilitated cell death rather than cell survival. Pan-caspase inhibition did not abrogate T-12 induced autophagy, suggesting that autophagy precedes apoptosis. In addition, T-12 inhibited cell survival pathway signaling proteins, Akt, mTOR and Erk1/2. T-12 also induced significant regression of tumor with oral dose of as low as 10 mg/kg bodyweight in rat mammary tumor model without any apparent toxicity. In presence of reactive oxygen species inhibitor (N-acetyl-l-cysteine) and autophagy inhibitor (chloroquine), T-12 induced tumor regression was significantly decreased. In conclusion, T-12 is a potent inducer of autophagy-dependent apoptosis in breast cancer cells both in vitro and in vivo and can serve as an important lead in development of new anti-tumor therapy.     

Oridonin up-regulates expression of P21 and induces autophagy and apoptosis in human prostate cancer cells

Background: Oridonin (ORI) could inhibit the proliferation and induce apoptosis in various cancer cell lines. However, the mechanism is not fully understood.Methods: Human prostate cancer (HPC) cells were cultured in vitro and cell viability was detected by the CCK-8 assay. The ultrastructure changes were observed under transmission electron microscope (TEM). Chemical staining with acridine orange (AO), MDC or DAPI was used to detect acidic vesicular organelles (AVOs) and alternation of DNA. Expression of LC3 and P21 was detected by Western Blot. Apoptotic rates and cell cycle arrest were detected by FACS.Results: Our study demonstrated that after ORI treatment, the proliferations of human prostate cancer (HPC) cell lines PC-3 and LNCaP were inhibited in a concentration and time-dependent manner. ORI induced cell cycle arrest at the G2/M phase. A large number of autophagosomes with double-membrane structure and acidic vesicular organelles (AVOs) were detected in the cytoplasm of HPC cells treated with ORI for 24 hours. ORI resulted in the conversion of LC3-I to LC3-II and recruitment of LC3-II to the autophagosomal membranes. Autophagy inhibitor 3-methyladenine (3-MA) reduced AVOs formation and inhibited LC3-I to LC3-II conversion. At 48 h, DNA fragmentation, chromatin condensation and disappearance of surface microvilli were detected in ORI-treated cells. ORI induced a significant increase in the number of apoptotic cells (PC-3: 5.4% to 27.0%, LNCaP: 5.3% to 31.0%). Promoting autophagy by nutrient starvation increased cell viability, while inhibition of autophagy by 3-MA promoted cell death. The expression of P21 was increased by ORI, which could be completely reversed by the inhibition of autophagy.Conclusions: Our findings indicated that autophagy occurred before the onset of apoptosis and protected cancer cells in ORI-treated HPC cells. P21 was involved in ORI-induced autophagy and apoptosis. Our results provide an experimental basis for understand the anti-tumor mechanism of ORI as treatment for prostate cancer.     

New Castanospermine Glycoside Analogues Inhibit Breast Cancer Cell Proliferation and Induce Apoptosis without Affecting Normal Cells

sp²-Iminosugar-type castanospermine analogues have been shown to exhibit anti-tumor activity. However, their effects on cell proliferation and apoptosis and the molecular mechanism at play are not fully understood. Here, we investigated the effect of two representatives, namely the pseudo-S- and C-octyl glycoside 2-oxa-3-oxocastanospermine derivatives SO-OCS and CO-OCS, on MCF-7 and MDA-MB-231 breast cancer and MCF-10A mammary normal cell lines. We found that SO-OCS and CO-OCS inhibited breast cancer cell viability in a concentration- and time-dependent manner. This effect is specific to breast cancer cells as both molecules had no impact on normal MCF-10A cell proliferation. Both drugs induced a cell cycle arrest. CO-OCS arrested cell cycle at G1 and G2/M in MCF-7 and MDA-MB-231cells respectively. In MCF-7 cells, the G1 arrest is associated with a reduction of CDK4 (cyclin-dependent kinase 4), cyclin D1 and cyclin E expression, pRb phosphorylation, and an overexpression of p21^Waf1/Cip1. In MDA-MB-231 cells, CO-OCS reduced CDK1 but not cyclin B1 expression. SO-OCS accumulated cells in G2/M in both cell lines and this blockade was accompanied by a decrease of CDK1, but not cyclin B1 expression. Furthermore, both drugs induced apoptosis as demonstrated by the increased percentage of annexin V positive cells and Bax/Bcl-2 ratio. Interestingly, in normal MCF-10A cells the two drugs failed to modify cell proliferation, cell cycle progression, cyclins, or CDKs expression. These results demonstrate that the effect of CO-OCS and SO-OCS is triggered by both cell cycle arrest and apoptosis, suggesting that these castanospermine analogues may constitute potential anti-cancer agents against breast cancer.     

Molecular evidence of anti-leukemia activity of gypenosides on human myeloid leukemia HL-60 cells in vitro and in vivo using a HL-60 cells murine xenograft model

We have shown that gypenosides (Gyp) induced cell cycle arrest and apoptosis in many human cancer cell lines. However, there are no reports showing that show Gyp acts on human leukemia HL-60 cells in vitro and in a murine xenograft model in vivo. In the present study effects of Gyp on cell morphological changes and viability, cell cycle arrest and induction of apoptosis in vitro and effects on Gyp in an in vivo murine xenograft model. Results indicated that Gyp induced morphological changes, decreased cell viability, induced G0/G1 arrest, DNA fragmentation and apoptosis (sub-G1 phase) in HL-60 cells. Gyp increased reactive oxygen species production and Ca²⁺ levels but reduced mitochondrial membrane potential in a dose- and time-dependent manner. Gyp also changed one of the primary indicators of endoplasmic reticulum (ER) stress due to the promotion of ATF6-α and ATF4-α associated with Ca²⁺ release. Gyp reduced the ratio of Bcl-2 to Bax due to an increase in the pro-apoptotic protein Bax and inhibited levels of the anti-apoptotic protein Bcl-2. Oral consumption of Gyp reduced tumor size of HL-60 cell xenograft mode mice in vivo. These results provide new information on understanding mechanisms by which Gyp induces cell cycle arrest and apoptosis in vitro and in vivo.     

Autophagy inhibition enhances ursolic acid-induced apoptosis in PC3 cells

The phosphoinositol 3-kinase/Akt pathway plays a critical role in oncogenesis and the dysregulation of this pathway through loss of PTEN is a particularly common phenomenon in aggressive prostate cancers. Several recent studies have indicated that ursolic acid (UA), a pentacyclic triterpenoid, and its derivatives inhibit the growth of cancer cells by cell cycle arrest and the stimulation of apoptosis. In the present study, we report a novel autophagic response of UA in PTEN-deficient PC3 prostate cancer cells. As one of the major types of programmed cell death, autophagy has been observed in response to several anticancer drugs and demonstrated to be responsible for cell death. UA-induced autophagy in PC3 cells is associated with the reduced cell viability and the enhanced expression of LC3-II, an autophagosome marker in mammals, and monodansylcadaverine incorporation into autolysosomes. Furthermore, we found that UA exhibited anti-proliferative effects characterized by G1 phase arrest and autophagy at an early stage that precedes apoptosis. We also show that UA-induced autophagy in PC3 cells are mediated through the Beclin-1 and Akt/mTOR pathways. Inhibition of autophagy by either 3-methyladenine or Beclin-1/Atg5 small interfering RNA enhanced UA-induced apoptosis. Taken together, our data suggest that autophagy functions as a survival mechanism in PC3 cells against UA-induced apoptosis and a rational for the use of autophagy inhibitors in combination with UA as a novel modality of cancer therapy.