Jolkinolide B induces cell cycle arrest and apoptosis in MKN45 gastric cancer cells and inhibits xenograft tumor growth in vivo

Gastric cancer is one of the most common digestive carcinomas throughout the world and represents high mortality. There is an urgent quest for seeking a novel and efficient antigastric cancer drug. Euphorbia fischeriana Steud had long been used as a traditional Chinese medicine for the treatment of cancer. According to the basic theory of traditional Chinese medicine, its antitumor mechanism is ‘to combat poison with poison’. However, its effective material foundation of it is still ambiguous. In our previous work, we studied the chemical constituents of E. fischeriana Steud. Jolkinolide B (JB) is an ent-abietane-type diterpenoid we isolated from it. The purpose of the present study was to investigate the antigastric effect and mechanism of JB. Results revealed that JB could suppress the proliferation of MKN45 cells in vitro and inhibit MKN45 xenograft tumor growth in nude mice in vivo. We further investigated its anticancer mechanism. On the one hand, JB caused DNA damage in gastric cancer MKN45 cells and induced the S cycle arrest by activating the ATR-CHK1-CDC25A-Cdk2 signaling pathway, On the other hand, JB induced MKN45 cells apoptosis through the mitochondrial pathway, and ultimately effectively inhibited the growth of gastric cancer cells. These results suggest that JB appears to be a promising candidate drug with antigastric cancer activity and warrants further research.     

A natural extracellular factor that induces Hsp72, inhibits apoptosis, and restores stress resistance in aged human cells

Experiments with cultured cells showed that most cellular stress resistance components are specialized for certain types of damage. For example, superoxide dismutase protects from oxidative damage; DNA repair enzymes guard against mutagens and other DNA-damaging agents. On the other hand, the major inducible heat shock protein Hsp72 protects cells from a large variety of stresses and thus represents a generalized repair/stress resistance component. Hsp72 not only refolds damaged proteins but also interferes with programmed cell death signaling pathways, thus providing cells with time to repair the damage, hence its universality as a stress protector. In the present study we demonstrate the occurrence in murine and human ascites fluids (AF) of a natural nontoxic extracellular factor (ascites Hsp72-inducing factor, AHIF) capable of activating Hsp72 expression in different types of cells via a pathway distinct from the heat shock response pathway. AHIF is unique in that it is the first physiological factor capable of inducing synthesis of Hsp72 not only in young cells but, remarkably, also in aged human cells that largely have lost the ability to express Hsp72 in response to stresses, a manifestation at the cellular level of a progressive impairment in the ability to adapt to environmental changes which characterizes aging. Pretreatment of aged human cells with AF triggers Hsp72 expression at levels seen in young stressed cells and protects cells from a variety of otherwise lethal stressful treatments such as heat shock, TNF, UV irradiation, etoposide, and menadione. Activation of Hsp72 expression is essential for antiapoptotic action of AHIF because specific inhibition of Hsp72 expression by antisense RNA abolishes the cytoprotective effect of AF. In view of an important link between stress resistance and longevity in different organisms, the abilities of AHIF make it a unique candidate for the role of a systemic regulator of the aging process. While a cell-autonomous stress response diminishes with aging, aged cells retain the ability to respond to an extracellular factor which induces the expression of Hsp72. This finding opens up exciting possibilities for using AF factor to restore stress resistance to old cells and organisms and the possibility of interfering with the aging process. The ability to induce stress resistance in young cells and to restore it in aged cells could serve as a basis for developing effective antiapoptotic therapies.     

Inhibition of NEDD4 inhibits cell growth and invasion and induces cell apoptosis in bladder cancer cells

The neural precursor cell expressed developmentally downregulated protein 4 (NEDD4) plays a pivotal oncogenic role in various types of human cancers. However, the function of NEDD4 in bladder cancer has not been fully investigated. In the present study, we aim to explore whether NEDD4 governs cell proliferation, apoptosis, migration, and invasion in bladder cancer cells. Our results showed that downregulation of NEDD4 suppressed cell proliferation in bladder cancer cells. Moreover, we found that inhibition of NEDD4 significantly induced cell apoptosis. Furthermore, downregulation of NEDD4 retarded cell migration and invasion. Notably, overexpression of NEDD4 enhanced cell growth and inhibited apoptosis. Consistently, upregulation of NEDD4 promoted cell migration and invasion in bladder cancer cells. Mechanically, our Western blotting results revealed that downregulation of NEDD4 activated PTEN and inhibited Notch-1 expression, whereas upregulation of NEDD4 reduced PTEN level and increased Notch-1 level in bladder cancer cells. Our findings indicated that NEDD4 exerts its oncogenic function partly due to regulation of PTEN and Notch-1 in bladder cancer cells. These results further revealed that targeting NEDD4 could be a useful approach for the treatment of bladder cancer.     

Novel levamisole derivative induces extrinsic pathway of apoptosis in cancer cells and inhibits tumor progression in mice

Background

Levamisole, an imidazo(2,1-b)thiazole derivative, has been reported to be a potential antitumor agent. In the present study, we have investigated the mechanism of action of one of the recently identified analogues, 4a (2-benzyl-6-(4′-fluorophenyl)-5-thiocyanato-imidazo[2,1-b][1], [3], [4]thiadiazole).

Materials and Methods

ROS production and expression of various apoptotic proteins were measured following 4a treatment in leukemia cell lines. Tumor animal models were used to evaluate the effect of 4a in comparison with Levamisole on progression of breast adenocarcinoma and survival. Immunohistochemistry and western blotting studies were performed to understand the mechanism of 4a action both ex vivo and in vivo.

Results

We have determined the IC₅₀ value of 4a in many leukemic and breast cancer cell lines and found CEM cells most sensitive (IC₅₀ 5 µM). Results showed that 4a treatment leads to the accumulation of ROS. Western blot analysis showed upregulation of pro-apoptotic proteins t-BID and BAX, upon treatment with 4a. Besides, dose-dependent activation of p53 along with FAS, FAS-L, and cleavage of CASPASE-8 suggest that it induces death receptor mediated apoptotic pathway in CEM cells. More importantly, we observed a reduction in tumor growth and significant increase in survival upon oral administration of 4a (20 mg/kg, six doses) in mice. In comparison, 4a was found to be more potent than its parental analogue Levamisole based on both ex vivo and in vivo studies. Further, immunohistochemistry and western blotting studies indicate that 4a treatment led to abrogation of tumor cell proliferation and activation of apoptosis by the extrinsic pathway even in animal models.

Conclusion

Thus, our results suggest that 4a could be used as a potent chemotherapeutic agent.     

Artocarpin induces cell apoptosis in human osteosarcoma cells through endoplasmic reticulum stress and reactive oxygen species

Osteosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. However, because of side effects and drug resistance in chemotherapy and the insufficiency of an effective adjuvant therapy for osteosarcoma, it is necessary to research novel treatments. This study was the first to investigate the anticancer effects of the flavonoid derivative artocarpin in osteosarcoma. Artocarpin induced cell apoptosis in three human osteosarcoma cell lines—U2OS, MG63, and HOS. Artocarpin was also associated with increased intracellular reactive oxygen species (ROS). Mitochondrial dysfunction was followed by the release of cytochrome c from mitochondria and accompanied by decreased antiapoptotic Bcl-2 and Bcl-xL and increased proapoptotic protein Bak and Bax. Artocarpin triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol calcium levels and increased glucose-regulated protein 78 and 94 expressions, and also increased calpains expression and activity. Animal studies revealed a dramatic 40% reduction in tumor volume after 18 days of treatment. This study demonstrated a novel anticancer activity of artocarpin against human osteosarcoma cells and in murine tumor models. In summary, artocarpin significantly induced cell apoptosis through ROS, ER stress, mitochondria, and the caspase pathway, and may thus be a novel anticancer treatment for osteosarcoma.     

siRNA targeting midkine inhibits gastric cancer cells growth and induces apoptosis involved caspase-3,8,9 activation and mitochondrial depolarization

Midkine (MK), a heparin-binding growth factor, is expressed highly in various malignant tumors, so it acts as attractive therapeutic target. In the present study, we used siRNA targeting MK to downregulate human MK expression in human gastric cancer cell line BGC823 and SGC7901 so as to determine the advantages of this anticancer therapeutic. The cell proliferation was evaluated by a WST-8 (4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate sodium salt) assay and colony formation assay. Apoptosis was determined by flow cytometer analysis and colorimetric assay. Our results showed that the BGC823 and SGC7901 cell growth were significantly inhibited by knockdown of MK gene. The loss of mitochondrial membrane potential, release of cytochrome c from the mitochondria into cytosol and increased activity of caspase-3, 8 and 9 occurred concomitantly with inhibition of MK gene. These results indicated that siRNA targeting MK gene can inhibit gastric cancer cells growth and induce apoptosis via mitochondrial depolarization and caspase-3 activation. MK siRNA may be a promising novel and potential therapeutic strategy for the treatment of gastric cancers.     

Salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells

Recently, salidroside (p-hydroxyphenethyl-β-d-glucoside) has been identified as one of the most potent compounds isolated from plants of the Rhodiola genus used widely in traditional Chinese medicine, but pharmacokinetic data on the compound are unavailable. We were the first to report the cytotoxic effects of salidroside on cancer cell lines derived from different tissues, and we found that human breast cancer MDA-MB-231 cells (estrogen receptor negative) were sensitive to the inhibitory action of low-concentration salidroside. To further investigate the cytotoxic effects of salidroside on breast cancer cells and reveal possible ER-related differences in response to salidroside, we used MDA-MB-231 cells and MCF-7 cells (estrogen receptor-positive) as models to study possible molecular mechanisms; we evaluated the effects of salidroside on cell growth characteristics, such as proliferation, cell cycle duration, and apoptosis, and on the expression of apoptosis-related molecules. Our results demonstrated for the first time that salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells and may be a promising candidate for breast cancer treatment.     

Naringenin inhibits migration,invasion, induces apoptosis in human lung cancer cells and arrests tumour progression in vitro

Lung cancer is one of the major cause for high-death rate all over the world, due to increased metastasize and difficulties in diagnosis. Naringenin is naturally occurring flavonoid found in various fruits including tomatoes, citrus fruit and figs. Naringenin is known to have several therapeutic effects including anti-atherogenic, antimicrobial, anti-inflammatory, hepatoprotective, anticancer and anti-mutagenic. The present study was aimed to analyse the naringenin induced anti-proliferative and apoptosis effects in human lung cancer cells. Cells were treated with various concentrations of naringenin (10, 100 & 200 µmol/L) for 48 hours. Cisplatin (20 µg/mL) was used as positive control. Cell viability, apoptosis, migration and mRNA, and protein expression of caspase-3, matrixmetallo proteinases-2 (MMP-2) and MMP-9 were determined. The cell viability was 93.7 ± 7.5, 51.4 ± 4.4 and 32.1 ± 2.1 at 10, 100 and 200 µmol/L of naringenin respectively. Naringenin significantly increased apoptotic cells. The 100 and 200 µmol/L of naringenin significantly suppressed the larger wounds of cultured human cancer cells compared with the untreated lung cancer cells. Naringenin increased d the expression of caspase-3 and reduced the expression of MMP-2 and MMP-9. Taking all these data together, it is suggested that the naringenin was effective against human lung cancer proliferation, migration and metastasis.     

Canstatin induces apoptosis in gastric cancer xenograft growth in mice through the mitochondrial apoptotic pathway

Canstatin, the non-collagenous domain of collagen type IV α-chains, belongs to a series of collagen-derived angiogenic inhibitors. In this study, the inhibitory effect of recombinant canstatin on tumour growth was investigated using a gastric cancer xenograft model. The volume and weight of tumours in mice treated with canstatin were lower than that in mice treated with PBS. Accordingly, the survival rate of these mice was significantly higher than that of mice bearing tumours treated with PBS. Moreover, valuable insight into the mechanisms mediated by canstatin was obtained.     

Methylglyoxal and high glucose co-treatment induces apoptosis or necrosis in human umbilical vein endothelial cells

Hyperglycemia and elevation of methylglyoxal (MG) are symptoms of diabetes mellitus (DM). We previously showed that high glucose (HG; 30 mM) or MG (50-400 microM) could induce apoptosis in mammalian cells, but these doses are higher than the physiological concentrations of glucose and MG in the plasma of DM patients. The physiological concentration of MG and glucose in the normal blood circulation is about 1 microM and 5 mM, respectively. Here, we show that co-treatment with concentrations of MG and glucose comparable to those seen in the blood circulation of DM patients (5 microM and 15-30 mM, respectively) could cause cell apoptosis or necrosis in human umbilical vein endothelial cells (HUVECs) in vitro. HG/MG co-treatment directly increased the reactive oxygen species (ROS) content in HUVECs, leading to increases in intracellular ATP levels, which can control cell death through apoptosis or necrosis. Co-treatment of HUVECs with 5 microM MG and 20 mM glucose significantly increased cytoplasmic free calcium levels, activation of nitric oxide synthase (NOS), caspase-3 and -9, cytochrome c release, and apoptotic cell death. In contrast, these apoptotic biochemical changes were not detected in HUVECs treated with 5 microM MG and 30 mM glucose, which appeared to undergo necrosis. Pretreatment with nitric oxide (NO) scavengers could inhibit 5 microM MG/20 mM glucose-induced cytochrome c release, decrease activation of caspase-9 and caspase-3, and increase the gene expression and protein levels of p53 and p21, which are known to be involved in apoptotic signaling. Inhibition of p53 protein expression using small interfering RNA (siRNA) blocked the activation of p21 and the cell apoptosis induced by 5 microM MG/20 mM glucose. In contrast, inhibition of p21 protein expression by siRNA prevented apoptosis in HUVECs but had no effect on p53 expression. These results collectively suggest that the treatment dosage of MG and glucose could determine the mode of cell death (apoptosis vs. necrosis) in HUVECs, and both ROS and NO played important roles in MG/HG-induced apoptosis of these cells.     

Linear ubiquitin chain induces apoptosis and inhibits tumor growth

Ubiquitination of proliferating cell nuclear antigen (PCNA) plays an important role in DNA damage response. Ectopic expression of PCNA fused at either terminus with ubiquitin (Ub) lacking two C-terminal glycine residues induces translesion DNA synthesis which resembles synthesis mediated by PCNA monoubiquitination. PCNA fused with Ub containing the C-terminal Gly residues at the C-terminus can be further polyubiquitinated in a Gly-dependent manner, which inhibits cell proliferation and induces ATR-dependent replication checkpoint. In this study, we surprisingly found that PCNA fused to a head-to-tail linear Ub chain induces apoptosis in a Ub chain length-dependent manner. Further investigation revealed that the apoptotic effect is actually induced by the linear Ub chain independently from PCNA, as the Ub chain fused to GFP or an epitope tag still efficiently induces apoptosis. It is revealed that the artificial linear Ub chain differs from endogenously encoded linear Ub chains in that its Ubs contain a Ub-G76S substitution, making the Ub chain resistant to cleavage by deubiquitination enzymes. We demonstrated in this study that ectopic expression of the artificial Ub chain alone in cultured human cancer cells is sufficient to inhibit tumor growth in a xenograft mouse model, making the linear Ub chain a putative anti-cancer agent.     

Guggulsterone induces apoptosis and inhibits lysosomal-dependent migration in human bladder cancer cells

BackgroundThe survival rate and therapeutic options for patients with bladder cancer have improved little in recent decades. Guggulsterone (GS), a phytoestrogen, has been investigated as an anticancer drug in various malignancies.PurposeThe present study aimed to evaluate the anticancer effects of E-isomer and Z-isomer GS in the human bladder cancer cell lines TSGH8301 (low-grade) and T24 (high-grade) and their underlying mechanisms.MethodsThe cell survival effect of GS was investigated by the MTT and colony formation assays in bladder cancer cell lines. Flow cytometry was used to analyze the cell cycle and cell death. Migration ability was measured by wound healing and transwell assays. Protein expression was determined by Western blot after GS treatment. The potency of GS on subcutaneous TSGH8301 bladder tumors was evaluated using an in vivo imaging system.ResultsE-isomer GS reduced the survival rate of both low- and high-grade human bladder cancer cells. GS caused cell cycle arrest, accompanied by the decrease and increase in cyclin A and p21 levels, respectively. Additionally, caspase-dependent apoptosis was observed following GS treatment. Furthermore, GS treatment downregulated mTOR-Akt signaling and induced autophagy with p62 and LC3β-II expression. Moreover, the farnesoid X receptor was involved in GS-inhibited cell growth. In addition, GS reduced the migration ability with a decrease in integrin-focal adhesion kinase and myosin light chain. Interestingly, the suppression of GS-mediated migration was prevented by the lysosomal inhibitor ammonium chloride (NH₄Cl). GS also reduced TSGH8301 bladder cancer cell progression by increasing the level of p21, cleaved caspase 3, cleaved poly (ADP-ribose) polymerase (PARP), and LC3β-II in vivo.ConclusionsThe current findings suggest that GS treatment may serve as a potential anticancer therapy for different grades of urothelial carcinoma.     

Adenosine induces apoptosis in human liver cancer cells through ROS production and mitochondrial dysfunction

Mitochondria are the most important sensor for apoptosis. Extracellular adenosine is well reported to induce apoptosis of tumor cells. Here we found that extracellular adenosine suppresses the cell growth by induction of apoptosis in BEL-7404 liver cancer cells, and identified a novel mechanism that extracellular adenosine triggers apoptosis by increasing Reactive Oxygen Species (ROS) production and mitochondrial membrane dysfunction in the cells. We observed that adenosine increases ROS production, activates c-Caspase-8 and -9 and Caspase effectors, c-Caspase-3 and c-PARP, induces accumulation of apoptosis regulator Bak, decreases Bcl-xL and Mcl-1, and causes the mitochondrial membrane dysfunction and the release of DIABLO, Cytochrome C, and AIF from mitochondria to cytoplasm in the cells; ROS inhibitor, NAC significantly reduces adenosine-induced ROS production; it also shows the same degree of blocking adenosine-induced loss of mitochondrial membrane potential (MMP) and apoptosis. Our study first observed that adenosine increases ROS production in tumor cells and identified the positive feedback loop for ROS-mediated mitochondrial membrane dysfunction which amplifies the death signals in the cells. Our findings indicated ROS production and mitochondrial dysfunction play a key role in adenosine-induced apoptosis of 7404 cells.     

Berberine inhibits human tongue squamous carcinoma cancer tumor growth in a murine xenograft model

Our primary studies showed that berberine induced apoptosis in human tongue cancer SCC-4 cells in vitro. But there is no report to show berberine inhibited SCC-4 cancer cells in vivo on a murine xenograft animal model. SCC-4 tumor cells were implanted into mice and groups of mice were treated with vehicle, berberine (10 mg/kg of body weight) and doxorubicin (4 mg/kg of body weight). The tested agents were injected once per four days intraperitoneally (i.p.), with treatment starting 4 weeks prior to cells inoculation. Treatment with 4 mg/kg of doxorubicin or with 10 mg/kg of berberine resulted in a reduction in tumor incidence. Tumor size in xenograft mice treated with 10 mg/kg berberine was significantly smaller than that in the control group. Our findings indicated that berbeirne inhibits tumor growth in a xenograft animal model. Therefore, berberine may represent a tongue cancer preventive agent and can be used in clinic.     

Telmisartan induces apoptosis and regulates Bcl-2 in human renal cancer cells

It has been well-characterized that the renin-angiotensin system (RAS) physiologically regulates systemic arterial pressure. However, RAS signaling has also been shown to increase cell proliferation during malignancy, and angiotensin receptor blockers (ARBs) are able to decrease pro-survival signaling by inhibiting anti-apoptotic molecules and suppressing caspase activity. In this study, the apoptotic effects of telmisartan, a type of ARB, was evaluated using a non-cancerous human renal cell line (HEK) and a human renal cell carcinoma (RCC) cell line (786). Both types of cells were treated with telmisartan for 4 h, 24 h, and 48 h, and then were assayed for levels of apoptosis, caspase-3, and Bcl-2 using MTT assays, flow cytometry, and immunostaining studies. Analysis of variance was used to identify significant differences between these data (P < 0.05). Following the treatment of 786 cells with 100 µM and 200 µM telmisartan, a marked inhibition of cell proliferation was observed. 50 µM cisplatin also caused high inhibition of these cells. Moreover, these inhibitions were both concentration- and time-dependent (P < 0.05). Various apoptotic effects were also observed compared with control cells at the 24 h and 48 h timepoints assayed (P < 0.001). Furthermore, positive caspase-3 staining and down-regulation of Bcl-2 were detected, consistent with induction of cell death. In contrast, treatment of HEK cells with telmisartan did not produce an apoptotic effect compared with control cells at the 24 h timepoint (P > 0.05). Treatment with cisplatin promoted in HEK cells high index of apoptosis (P < 0.001). Taken together, these results suggest that telmisartan induces apoptosis via down-regulation of Bcl-2 and involvement of caspase-3 in human RCC cells.     

Dihydroartemisinin induces apoptosis and sensitizes human ovarian cancer cells to carboplatin therapy

The present study was designed to determine the effects of artemisinin (ARS) and its derivatives on human ovarian cancer cells, to evaluate their potential as novel chemotherapeutic agents used alone or in combination with a conventional cancer chemotherapeutic agent, and to investigate their underlying mechanisms of action. Human ovarian cancer cells (A2780 and OVCAR-3), and immortalized non-tumourigenic human ovarian surface epithelial cells (IOSE144), were exposed to four ARS compounds for cytotoxicity testing. The in vitro and in vivo antitumour effects and possible underlying mechanisms of action of dihydroartemisinin (DHA), the most effective compound, were further determined in ovarian cancer cells. ARS compounds exerted potent cytotoxicity to human ovarian carcinoma cells, with minimal effects on non-tumourigenic ovarian surface epithelial (OSE) cells. DHA inhibited ovarian cancer cell growth when administered alone or in combination with carboplatin, presumably through the death receptor- and, mitochondrion-mediated caspase-dependent apoptotic pathway. These effects were also observed in in vivo ovarian A2780 and OVCAR-3 xenograft tumour models. In conclusion, ARS derivatives, particularly DHA, exhibit significant anticancer activity against ovarian cancer cells in vitro and in vivo , with minimal toxicity to non-tumourigenic human OSE cells, indicating that they may be promising therapeutic agents for ovarian cancer, either used alone or in combination with conventional chemotherapy.     

The protozoan toxin climacostol inhibits growth and induces apoptosis of human tumor cell lines

Climacostol (5-(Z)-non-2-enyl-benzene-1,3-diol) is a natural toxin isolated from the freshwater ciliated protozoan Climacostomum virens and belongs to the group of resorcinolic lipids, compounds that show antimicrobial, antiparasitic and antitumor activities. We investigated the cytotoxic activity of the chemically synthesized toxin on: (1) human tumor squamous carcinoma A431 cells, (2) human promyelocytic leukaemia HL60 cells, and (3) human non-tumor endothelial EA.hy926 cells. The results showed that climacostol effectively inhibited the growth of tumor cell lines in a dose-dependent manner by inducing programmed cell death, with non-tumor cells proving significantly more resistant to the toxin.