Cancer is a multistep process resulting in uncontrolled cell division. It results from aberrant signaling pathways that lead to uninhibited cell division and growth. Various recent epidemiological studies have indicated that consumption of cruciferous vegetables such as garden cress, broccoli, etc., reduces the risk of cancer. Isothiocyanates (ITC) have been identified as major active constituents of cruciferous vegetables. ITCs occur in plants as glucosinolate and can readily be derived by hydrolysis. Numerous mechanistic studies have demonstrated the anti-cancer effects of ITCs in various cancer types. ITCs suppress tumor growth by generating reactive oxygen species or by inducing cycle arrest leading to apoptosis. Based on the exciting outcomes of pre-clinical studies, few ITCs have advanced to the clinical phase. Available data from pre-clinical as well as available clinical studies suggests ITCs to be one of the promising anti-cancer agents available from natural sources. This is an up-to-date exhaustive review on the preventive and therapeutic effects of ITCs in cancer.
BITC; PEITC; Sulforaphane; AITC; Isothiocyanate; cancer
Aberrant activation of β-catenin/TCF signaling is related to the invasiveness of pancreatic cancer. In the present study, we evaluated the effect of capsaicin on β-catenin/TCF signaling. In a concentration and time-dependent study, we observed that capsaicin treatment inhibits the activation of dishevelled (Dsh) protein DvI-1 in L3.6PL, PanC-1 and MiaPaCa-2 pancreatic cancer cells. Capsaicin treatment induced GSK-3β by inhibiting its phosphorylation and further activated APC and Axin multicomplex, leading to the proteasomal degradation of β-catenin. Expression of TCF-1 and β-catenin-responsive proteins, c-Myc and cyclin D1 also decreased in response to capsaicin treatment. Pre-treatment of cells with MG-132 blocked capsaicin-mediated proteasomal degradation of β-catenin. To establish the involvement of β-catenin in capsaicin-induced apoptosis, cells were treated with LiCl or SB415286, inhibitors of GSK-3β. Our results reveal that capsaicin treatment suppressed LiCl or SB415286-mediated activation of β-catenin signaling. Our results further showed that capsaicin blocked nuclear translocation of β-catenin, TCF-1 and p-STAT-3 (Tyr705). The immunoprecipitation results indicated that capsaicin treatment reduced the interaction of β-catenin and TCF-1 in the nucleus. Moreover, capsaicin treatment significantly decreased the phosphorylation of STAT-3 at Tyr705. Interestingly, STAT-3 over expression or STAT-3 activation by IL-6, significantly increased the levels of β-catenin and attenuated the effects of capsaicin in inhibiting β-catenin signaling. Finally, capsaicin mediated inhibition of orthotopic tumor growth was associated with inhibition of β-catenin/TCF-1 signaling. Taken together, our results suggest that capsaicin-induced apoptosis in pancreatic cancer cells was associated with inhibition of β-catenin signaling due to the dissociation of β-catenin/TCF-1 complex and the process was orchestrated by STAT-3.
STAT3; β-Catenin; GSK-3β; pancreatic cancer; orthotopic tumor
Anoikis is an anchorage-independent cell death. Resistance to anoikis is one of the key features of metastatic cells. Here, we analyzed the role of STAT3 in anoikis resistance in melanoma cells leading to metastasis. When grown under anchorage-independent conditions, significant proportion of cells resisted anoikis and these resistant cells had higher rate of migration and invasion as compared to the cells grown under anchorage-dependent conditions. The anoikis resistant cells also had significantly higher expression and phosphorylation of STAT3 at Y705 than the cells that were attached to the basement membrane. STAT3 inhibitors, AG 490 and piplartine (PL) induced anoikis in a concentration-dependent manner in anoikis resistant cells. Over-expression of STAT3 or treatment with IL-6 not only increased anoikis resistance, but also protected the cancer cells from PL-induced anoikis. On the other hand, silencing STAT3 decreased the potential of cancer cells to resist anoikis and to migrate. STAT3 knock-down cells and PL treated cells did not form tumors as well as failed to metastasize in SCID-NSG mice as compared to untreated anchorage-independent cells, which formed big tumors and extensively metastasized. In summary, our results for the first time establish STAT3 as a critical player that renders anoikis resistance to melanoma cells and enhance their metastatic potential.
STAT3; anoikis resistance; metastasis; melanoma
Pancreatic cancer is fourth leading cause of cancer-related deaths in the United States of America. In spite of recent advances in the current therapeutic modalities such as surgery, radiation and chemotherapy patients, the average five year survival rate remains still less than 5%. Recently, compounds from natural sources receive ample of attention as anti-cancer agents. Many epidemiological studies published over the past few decades provide a strong correlation between consumption of vegetables, fruits or plant derived products and reduced incidence of cancer. The present review focuses on the potential antitumor effects of various natural products.
Benzyl isothiocyanate; Capsaicin; Resveratrol; Green tea; Curcumin
HER2, an oncogenic receptor is overexpressed in about 25-30% of breast cancer patients. HER2 has been shown to play role in tumor promotion by having cross-talk with multiple oncogenic pathways in cancer cells. Our results show that Cucurbitacin B (CuB), a triterpenoid steroidal compound inhibited the growth of various breast cancer cells with an IC50 ranging from 18-50nM after 48 and 72 h of treatment. Our study also revealed the significant inhibitory effects of CuB on HER2 and integrin signaling in breast cancer. Notably, CuB inhibited ITGA6 and ITGB4 (integrin α6 & integrin β4), which are overexpressed in breast cancer. Furthermore, CuB also induced the expression of major ITGB1and ITGB3, which are known to cause integrin-mediated cell death. In addition, we observed that TGFβ treatment resulted in the increased association of HER2 with ITGA6 and this association was inhibited by CuB treatment. Efficacy of CuB was tested in vivo using two different orthotopic models of breast cancer. MDA-MB-231 and 4T-1 cells were injected orthotopically in the mammary fat pad of female athymic nude mice or BALB/c mice respectively. Our results showed that CuB administration inhibited MDA-MB-231 orthotopic tumors by 55%, and 4T-1 tumors by 40%. The 4T-1 cells represent stage IV breast cancer and form very aggressive tumors. CuB mediated breast tumor growth suppression was associated with the inhibition of HER2/integrin signaling. Our results suggest novel targets of CuB in breast cancer in vitro and in vivo.
Breast cancer; in vivo; ITGB4; ITGA6; Cucurbitacin B
Deguelin is known to suppress the growth of cancer cells; however, its anti-metastatic effects have not been studied so far in any cancer model. In the present study, we aimed to evaluate the anti-metastatic potential of deguelin in vivo and in TGFβ1-stimulated cells. Our results demonstrate that tumor growth, peritoneal-dissemination and liver/lung metastasis of orthotopically implanted PanC-1-luc cells were significantly reduced in deguelin-treated mice along with the induction of apoptosis. Furthermore, deguelin-treated tumors showed increased epithelial signature such as increased expression of E-Cadherin and cytokeratin-18 and decreased expression of Snail. Similar observations were made when PanC-1, COLO-357 and L3.6pl cells were treated in vitro with deguelin. Moreover, E-cadherin was transcriptionally up-regulated and accumulated in the membrane fraction of deguelin-treated cells as indicated by increased interaction of E-Cadherin with β-catenin. TGFβ1-induced down-regulation of E-Cadherin and up-regulation of Snail were abrogated by deguelin treatment. In addition, deguelin inhibited TGFβ1-induced Smad3 phosphorylation and Smad4 nuclear translocation in PanC-1 cells. Furthermore, when TGFβ1-induced NFkB activation was inhibited, TGFβ1-induced Snail up-regulation or E-Cadherin down-regulation was blocked. Deguelin also significantly down regulated the constitutive phosphorylation and DNA binding of NFkB in a dose dependent manner. Interestingly, overexpression of either NFkB or Snail completely abrogated deguelin-mediated EMT inhibition, whereas overexpression of NFkB but not Snail rescued cells from deguelin-induced apoptosis. Hence, deguelin targets NFkB to induce reversal of EMT and apoptosis but downstream effectors might be different for both processes. Taken together, our results suggest that deguelin suppresses both pancreatic tumor growth and metastasis by inducing apoptosis and inhibiting epithelial to mesenchymal transition.
NFkB; TGF-β; RKIP; Snail; Metastasis; Apoptosis
Aim: In this study, we evaluated the effect of capsaicin on the interaction of redox-sensitive thioredoxin (Trx)/apoptosis signal-regulating kinase 1 (ASK1) in pancreatic cancer cells. Results: Capsaicin treatment downregulated Trx and increased the phosphorylation (activation) of ASK1 at Thr845 and kinase activity in AsPC-1 and BxPC-3 cells. Capsaicin treatment also activated downstream effector molecules MKK4/7, caspase-9, and caspase-3. Antioxidants tiron or PEG-catalase blocked the activation of ASK1 cascade by capsaicin and protected the cells from apoptosis, indicating the involvement of reactive oxygen species in the activation of ASK1. Our results further revealed that Trx overexpression suppressed the effects of capsaicin, whereas ASK1 overexpression enhanced the apoptosis-inducing effects of capsaicin. β-mercaptoethanol, a reducing agent, blocked capsaicin-mediated activation of ASK1, indicating that Trx-ASK1 complex exists and requires reducing conditions in the cell. On the other hand, the Trx inhibitor (1-chloro-2-4-dinitrobenzene) increased capsaicin-induced ASK1 kinase activity, suggesting that Trx inhibition by capsaicin is essential for ASK1 activation. Oral administration of 5 mg capsaicin/kg body weight substantially suppressed the growth of tumors in xenograft and orthotopic mouse model. Tumors from capsaicin-treated mice showed reduced levels of Trx, increased phosphorylation of ASK1 at Thr845, and cleavage of caspase-3 and poly (ADP-ribose) polymerase. Innovation: Our results for the first time demonstrated a new perspective that Trx-ASK1 complex can be targeted by capsaicin in pancreatic cancer. Conclusion: Capsaicin reduces Trx expression and dissociates Trx-ASK1 complex resulting in the activation of ASK1 and downstream effectors leading to apoptosis in pancreatic tumor cells in vitro and in vivo. Antioxid. Redox Signal. 17, 1417–1432.
Capsaicin is a major biologically active ingredient of chili peppers. Extensive studies indicate that capsaicin is a cancer-suppressing agent via blocking the activities of several signal transduction pathways including nuclear factor-kappaB, activator protein-1 and signal transducer and activator of transcription 3. However, there is little study on the effect of capsaicin on pancreatic carcinogenesis. In the present study, the effect of capsaicin on pancreatitis and pancreatic intraepithelial neoplasia (PanIN) was determined in a mutant Kras-driven and caerulein-induced pancreatitis-associated carcinogenesis in LSL-KrasG12D/Pdx1-Cre mice. Forty-five LSL-KrasG12D/Pdx1-Cre mice and 10 wild-type mice were subjected to one dose of caerulein (250 μg/kg body wt, intraperitoneally) at age 4 weeks to induce and synchronize the development of chronic pancreatitis and PanIN lesions. One week after caerulein induction, animals were randomly distributed into three groups and fed with either AIN-76A diet, AIN-76A diet containing 10 p.p.m. capsaicin or 20 p.p.m. capsaicin for a total of 8 weeks. The results showed that capsaicin significantly reduced the severity of chronic pancreatitis, as determined by evaluating the loss of acini, inflammatory cell infiltration and stromal fibrosis. PanIN formation was frequently observed in the LSL-KrasG12D/Pdx1-Cre mice. The progression of PanIN-1 to high-grade PanIN-2 and -3 were significantly inhibited by capsaicin. Further immunochemical studies revealed that treatment with 10 and 20 p.p.m. capsaicin significantly reduced proliferating cell nuclear antigen-labeled cell proliferation and suppressed phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun as well blocked Hedgehog/GLI pathway activation. These results indicate that capsaicin could be a promising agent for the chemoprevention of pancreatic carcinogenesis, possibly via inhibiting pancreatitis and mutant Kras-led ERK activation.
Our previous studies have shown that benzyl isothiocyante (BITC) suppresses pancreatic cancer growth by inducing apoptosis but the molecular mechanism was unclear. In the present study we hypothesized the involvement of PI3K/AKT/FOXO pathway in BITC induced apoptosis.
Mice were implanted BxPC-3 tumor xenografts and orally gavaged with 12μmol BITC. Plasma and tumor BITC concentration was estimated by LC/MS/MS. BxPC-3 and PanC-1 cells were used to elucidate PI3K/AKT/FOXO pathway. EMSA, DNA binding activity, immunofluorescence and gene transfection were used to delineate the mechanism.
BITC-treated mice showed 43% less tumor growth as compared to control mice and correlated well with the therapeutic concentrations of 6.5μM BITC achieved in plasma and 7.5μmol/g BITC in tumor tissue. Western blot analyses and immunohistochemistry revealed that tumors from BITC-treated mice demonstrated reduced phosphorylation of PI3K, AKT, PDK1, mTOR, FOXO1, FOXO3a and increased apoptosis. Complementing our in vivo results, we made similar observations in a dose and time-dependent manner in BITC-treated BxPC-3 and Panc-1 cells. Binding of FOXO1 with 14-3-3 proteins was also reduced drastically by BITC treatment indicating nuclear retention of FOXO1 and this observation was further confirmed with EMSA, immunofluorescence, DNA binding and up regulation of FOXO-responsive proteins Bim, p27 and p21 in BxPC-3 cells. Overexpression of AKT by transient transfection significantly blocked the modulation of FOXO proteins and protected the cells from BITC mediated apoptosis and growth suppression.
Our results provide convincing evidence on the involvement of PI3K/AKT/FOXO pathway in BITC mediated pancreatic tumor growth suppression.
Benzyl isothiocyanate; AKT; FOXO; Pancreatic cancer; chemoprevention
In this study we show that diindolylmethane (DIM) induces autophagy in ovarian cancer cells by regulating endoplasmic reticulum (ER) stress and AMPK. Treatment of SKOV-3, OVCAR-3 and TOV-21G ovarian cancer cells with varying concentrations of DIM for 24 hours resulted in a concentration dependent induction of autophagy as measured by flowcytometry. Electron microscopy confirmed the presence of autophagosomes in DIM treated cells. Western blot analysis showed that DIM treatment increased the expression of LC3B, a hall mark of autophagy as well as p62 and Atg 12 proteins that are accumulated during autophagy. Autophagy inhibitors bafilomycin or chloroquine inhibited DIM induced autophagy. Furthermore, DIM treatment significantly increased the expression of ER stress regulators such as Grp78, IRE1 and GADD153. Cycloheximide or ER stress inhibitor mithramycin not only blocked ER stress proteins that were activated by DIM but also autophagy. Silencing Grp78 or GADD 153 significantly blocked the expression of LC3B and p62 indicating that autophagy in our model is mediated by ER stress. Knocking out LC3B inhibited DIM induced autophagy. DIM treatment increased the cytosolic calcium levels which lead to the activation of AMPK in our model. Chelating cytosolic calcium with BAPT-AM abrogated not only the phosphorylation of AMPK but also prevented DIM induced autophagy. Inhibiting AMPK by a chemical inhibitor or siRNA blocked the induction of LC3B or p62, indicating that DIM mediated autophagy requires activation of AMPK. Oral administration of DIM significantly suppressed SKOV-3 tumor xenografts in nude mice. Activation of ER stress and autophagy were observed in the tumors of DIM treated mice. Taken together, these results suggest that induction of autophagy by DIM in ovarian cancer cells was associated with ER stress and AMPK activation.
Diindolylmethane; ER stress; autophagy; apoptosis
NF-κB/p65 is constitutively activated in pancreatic cancers where it plays critical role in the transcriptional activation of multiple cell survival genes. We have previously demonstrated the apoptosis-inducing effects of BITC in pancreatic cancer cells. We hypothesized that inhibition of NF-κB/p65 could be the mechanism of BITC-induced apoptosis. Therefore, the effect of BITC on NF-κB/p65 was evaluated in BxPC-3, Capan-2 and normal HPDE-6 cells by western blotting, transcriptional and DNA-binding activity and by immunohistochemistry in the xenografted tumors. Our results reveal a remarkable decrease in the phosphorylation of NF-κB/p65 at Ser536 in both BxPC-3 and Capan-2 cells by BITC treatment. The expression of NF-kB/p65 was down-regulated significantly in BxPC-3 cells whereas it remained unchanged in Capan-2 cells. BITC treatment caused significant decrease in NF-κB transcriptional and DNA-binding activity in both BxPC-3 and Capan-2 cells. A drastic decrease was observed in the expression and reporter activity of cyclin D1 in both the cell lines. Moreover, BITC also caused significant decrease in the expression and activity of HDAC1 and HDAC3 in BxPC-3 and HDAC3 in Capan-2 cells. Overexpression of HDAC1 or HDAC3 abrogated the effects of BITC. BITC treatment did not caused any change in HDAC expression in normal HPDE-6 cells. Immunohistochemical analysis of tumors from BITC-treated mice showed significantly reduced staining for NF-kB, cyclin D1, HDAC-1/3, compared to control. Our results suggest that inhibition of HDAC1/3 by BITC as a plausible mechanism of NF-κB inactivation resulting in the in vitro and in vivo growth suppression of pancreatic cancer cells.
Benzyl isothiocyanate; NF-kB; HDAC-3; HDAC-1; Cyclin D1; pancreatic cancer chemoprevention
Benzyl isothiocyanate (BITC), a compound found in cruciferous vegetables, has been reported to have anticancer properties, but the mechanism whereby it inhibits growth of human pancreatic cancer cells is incompletely understood.
Human pancreatic cancer cells (BxPC-3, AsPC-1, Capan-2, MiaPaCa-2, and Panc-1) and immortalized human pancreatic cells (HPDE-6) were treated with vehicle or with BITC at 5–40 μM, cell survival was evaluated by sulforhodamine B assay, and apoptosis by caspase-3 and poly-ADP ribose polymerase cleavage or by a commercial assay for cell death. Total and activated signal transducer and activator of transcription-3 (STAT-3) protein expression in the cells were examined by western blotting, STAT-3 mRNA levels by reverse transcription–polymerase chain reaction, and STAT-3 DNA-binding and transcriptional activity by commercially available binding and reporter assays. The effects of BITC treatment on tumor growth, apoptosis, and STAT-3 protein expression in vivo were studied in xenografts of BxPC-3 pancreatic tumor cells in athymic nude mice. All statistical tests were two-sided.
BITC treatment reduced cell survival and induced apoptosis in BxPC-3, AsPC-1, Capan-2, and MiaPaCa-2 cells, and to a much lesser extent in Panc-1 cells, but not in HPDE-6 cells. It also reduced levels of activated and total STAT-3 protein, and as a result, STAT-3 DNA-binding and transcriptional activities. Overexpression of STAT-3 in BxPC-3 cells inhibited BITC-induced apoptosis and restored STAT-3 activity. In mice that were fed BITC (60 μmol/wk, five mice, 10 tumors per group), growth of BxPC-3 pancreatic tumor xenografts was suppressed compared with control mice (at 6 weeks, mean tumor volume of control vs BITC-treated mice = 334 vs 172 mm3, difference =162 mm3, 95% confidence interval = 118 to 204 mm3; P = .008) and tumors had increased apoptosis and reduced STAT-3 protein expression.
BITC induces apoptosis in some types of pancreatic cancer cells by inhibiting the STAT-3 signaling pathway.
A new series of fifteen 5-, 6-, and 8-appended 4-methylquinolines were synthesized and evaluated for their neural protective activities. Selected compounds were further examined for their inhibition of glycogen synthase kinase-3β (GSK-3β) and protein kinase C (PKC). Two most potent analogs, compounds 3 and 10, show nanomolar protective activities in amyloid β-induced MC65 cells and enzymatic inhibitory activities against GSK-3β, but poor PKC inhibitory activities. Using normal mouse model, the distribution of the most potent analog 3 in various tissues and possible toxic effects in the locomotors and inhibition of liver transaminases activities were carried out. No apparent decline of locomotor activity and no inhibition of liver transaminases were found. The compound appears to be safe for long-term use in Alzheimer's disease mouse model.
Alzheimer's disease; amyloid β; glycogen synthase kinase-3β; neural protective activity; protein kinase C; quinolines
Here we investigated the potential mechanism of capsaicin-mediated apoptosis in pancreatic cancer cells. Capsaicin treatment phosphorylated JNK, FOXO1 and BIM in BxPC-3, AsPC-1 and L3.6PL cells. The expression of BIM increased in response to capsaicin treatment. Capsaicin treatment caused cleavage of caspase-3and PARP indicating apoptosis. Antioxidants tiron and PEG-catalase blocked capsaicin-mediated JNK/FOXO/BIM activation and protected the cells from apoptosis. Furthermore, capsaicin treatment caused a steady increase in the nuclear expression of FOXO-1 leading to increased DNA binding. Capsaicin-mediated expression of BIM was found to be directly dependent on the acetylation of FOXO-1. The expression of CBP was increased whereas SirT-1was reduced by capsaicin treatment. Using acetylation mimic or defective mutants, our result demonstrated that phosphorylation of FOXO-1 was mediated through acetylation by capsaicin treatment. JNK inhibitor attenuated the phosphorylation of FOXO-1, activation of BIM and abrogated capsaicin-induced apoptosis. Moreover, silencing FOXO1 by siRNA blocked capsaicin-mediated activation of BIM and apoptosis whereas overexpression of FOXO-1augmented its effects. Silencing Bim drastically reduced capsaicin-mediated cleavage of caspase-3 and PARP, indicating the role of BIM in apoptosis. Oral administration of 5mg/kg capsaicin substantially suppressed the growth of BxPC-3 tumor xenografts in athymic nude mice. Tumors from capsaicin-treated mice showed an increase in the phosphorylation of JNK, FOXO-1, BIM, and levels of CBP, cleavage of caspase-3, PARP and decreased SirT-1 expression. Taken together, our results suggest that capsaicin activated JNK and FOXO-1, leading to the acetylation of FOXO-1 through CBP and SirT-1. Acetylated FOXO1 induced apoptosis in pancreatic cancer cells through BIM activation.
Pancreatic cancer; Capsaicin; Apoptosis; Bim; FOXO-1 acetylation
Melanoma is highly metastatic and resistant to chemotherapeutic drugs. Our previous studies have demonstrated that caffeic acid phenethyl ester (CAPE) suppresses the growth of melanoma cells and induces reactive oxygen species generation. However, the exact mechanism of the growth suppressive effects of CAPE was not clear. Here, we determined the potential mechanism of CAPE against melanoma in vivo and in vitro. Administration of 10 mg/kg/day CAPE substantially suppressed the growth of B16F0 tumor xenografts in C57BL/6 mice. Tumors from CAPE-treated mice showed reduced phosphorylation of phosphoinositide 3-kinase, AKT, mammalian target of rapamycin and protein level of X-linked inhibitor of apoptosis protein (XIAP) and enhanced the cleavage of caspase-3 and poly (ADP ribose) polymerase. In order to confirm the in vivo observations, melanoma cells were treated with CAPE. CAPE treatment suppressed the activating phosphorylation of phosphoinositide 3-kinase at Tyr 458, phosphoinositide-dependent kinase-1 at Ser 241, mammalian target of rapamycin at Ser 2448 and AKT at Ser 473 in B16F0 and SK-MEL-28 cells in a concentration and time-dependent study. Furthermore, the expression of XIAP, survivin and BCL-2 was downregulated by CAPE treatment in both cell lines. Significant apoptosis was observed by CAPE treatment as indicated by cleavage of caspase-3 and poly (ADP ribose) polymerase. AKT kinase activity was inhibited by CAPE in a concentration-dependent manner. CAPE treatment increased the nuclear translocation of XIAP, indicating increased apoptosis in melanoma cells. To confirm the involvement of reactive oxygen species in the inhibition of AKT/XIAP pathway, cells were treated with antioxidant N-acetyl-cysteine (NAC) prior to CAPE treatment. Our results indicate that NAC blocked CAPE-mediated AKT/XIAP inhibition and protected the cells from apoptosis. Because AKT regulates XIAP, their interaction was examined by immunoprecipitation studies. Our results show that CAPE treatment decreased the interaction of AKT with XIAP. To establish the involvement of AKT in the apoptosis-inducing effects of CAPE, cells were transfected with AKT. Our results revealed that AKT overexpression attenuated the decrease in XIAP and significantly blocked CAPE-mediated apoptosis. Similarly, overexpression of XIAP further decreased CAPE-induced apoptosis. Taken together, our results suggest that CAPE suppresses phosphoinositide 3-kinase/AKT/XIAP pathway leading to apoptosis in melanoma tumor cells in vitro and in vivo.
In this study, we determined the cytotoxic effects of piperine, a major constituent of black and long pepper in melanoma cells. Piperine treatment inhibited the growth of SK MEL 28 and B16 F0 cells in a dose and time-dependent manner. The growth inhibitory effects of piperine were mediated by cell cycle arrest of both the cell lines in G1 phase. The G1 arrest by piperine correlated with the down-regulation of cyclin D1 and induction of p21. Furthermore, this growth arrest by piperine treatment was associated with DNA damage as indicated by phosphorylation of H2AX at Ser139, activation of ataxia telangiectasia and rad3-related protein (ATR) and checkpoint kinase 1 (Chk1). Pretreatment with AZD 7762, a Chk1 inhibitor not only abrogated the activation of Chk1 but also piperine mediated G1 arrest. Similarly, transfection of cells with Chk1 siRNA completely protected the cells from G1 arrest induced by piperine. Piperine treatment caused down-regulation of E2F1 and phosphorylation of retinoblastoma protein (Rb). Apoptosis induced by piperine was associated with down-regulation of XIAP, Bid (full length) and cleavage of Caspase-3 and PARP. Furthermore, our results showed that piperine treatment generated ROS in melanoma cells. Blocking ROS by tiron protected the cells from piperine mediated cell cycle arrest and apoptosis. These results suggest that piperine mediated ROS played a critical role in inducing DNA damage and activation of Chk1 leading to G1 cell cycle arrest and apoptosis.
The asymmetric unit of the title compound, C18H15ClTe, contains two molecules which are in inverted orientations. The compound displays a tetrahedral geometry around the Te atom in spite of there being five electron domains. This is attributed to the fact that the lone pair is not sterically active. The dihedral angles between the three phenyl rings are 76.51 (16)/73.75 (16)/71.06 (17) and 78.60 (17)/77.67 (16)/79.11 (16)° in the two molecules. The crystal packing features eight C—H⋯π interactions.
The aim of the present study was to determine whether the combination of two modalities of immunotherapy, targeting two different tumor antigens, may be feasible and non-toxic, yet enhance the killing of a human breast cancer cell line. The first modality was tumor growth factor α-Pseudomonas exotoxin 38 (TGFα-PE38), which specifically targets and kills tumor cells that express the epidermal growth factor receptor. The second modality was mucin-1 (MUC1)-specific cytotoxic T lymphocytes (CTLs), generated by MUC1 stimulation of peripheral blood mononuclear cells, to target the human breast cancer cell line, MCF7. TGFα-PE38 exhibited specific lysis of the MCF7 cells in a concentration- and time-dependent manner. TGFα-PE38 did not kill the normal hematopoietic stem cells or CTLs. Furthermore, TGFα-PE38 was not inhibitory for the growth or differentiation of the normal human hematopoietic stem cells into erythroid and myeloid colonies. In addition, TGFα-PE38 did not inhibit the killing function of CTLs, either when preincubated or co-incubated with CTLs. Finally, therapeutic enhancement was observed, in that TGFα-PE38 and CTLs were additive in the specific lysis of the MCF7 cells. These two modalities of immunotherapy may be beneficial for humans with breast cancer with or without other therapies, including autologous hematopoietic stem cell transplantation, specifically for purging cancer cells from hematopoietic stem cells prior to transplantation.
TGFα-PE38; cytotoxic T lymphocyte; cancer; breast; mucin-1
This study characterized electrophilic and radical products derived from metabolism of capsaicin by cytochrome P450 and peroxidase enzymes. Multiple glutathione and β-mercaptoethanol conjugates (a.k.a., adducts), derived from trapping of quinone methide and quinone intermediates of capsaicin, its analogue nonivamide, and O-demethylated and aromatic hydroxylated metabolites thereof, were produced by human liver microsomes and individual recombinant human P450 enzymes. Conjugates derived from concomitant dehydrogenation of the alkyl terminus of capsaicin, were also characterized. Modifications to the 4-OH substituent of the vanilloid ring of capsaicinoids largely prevented the formation of electrophilic intermediates, consistent with the proposed structures and mechanisms of formation for the various conjugates. 5,5’-Dicapsaicin, presumably arising from bi-molecular coupling of free radical intermediates, was also characterized. Finally, the analysis of hepatic glutathione conjugates and urinary N-acetylcysteine conjugates from mice dosed with capsaicin confirmed the formation of glutathione conjugates of O-demethylated, quinone methide, and 5-OH-capsaicin in vivo. These data demonstrated that capsaicin and structurally similar analogues are converted to reactive intermediates by certain P450 enzymes, which may partially explain conflicting reports related to the cytotoxic, pro-carcinogenic, and chemoprotective effects of capsaicinoids in different cells and/or organ systems.
Capsaicin; nonivamide; cytochrome P450; electrophile; glutathione conjugate
Breast tumor metastasis is a leading cause of cancer-related deaths worldwide. Breast tumor cells frequently metastasize to brain and initiate severe therapeutic complications. The chances of brain metastasis are further elevated in patients with HER2 overexpression. In the current study, we evaluated the anti-metastatic effects of phenethyl isothiocyanate (PEITC) in a novel murine model of breast tumor metastasis. The MDA-MB-231-BR (BR-brain seeking) breast tumor cells stably transfected with luciferase were injected into the left ventricle of mouse heart and the migration of cells to brain was monitored using a non-invasive IVIS bio-luminescent imaging system. In order to study the efficacy of PEITC in preventing the number of tumor cells migrating to brain, mice were given 10 µmol PEITC by oral gavage for ten days prior to intra-cardiac injection of tumor cells labeled with quantum dots. To evaluate the tumor growth suppressive effects, 10 µmol PEITC was given to mice every day starting 14th day after intra-cardiac cell injection. Based on the presence of quantum dots in the brain section of control and treated mice, our results reveal that PEITC significantly prevented the metastasis of breast cancer cells to brain. Our results demonstrate that the growth of metastatic brain tumors in PEITC treated mice was about 50% less than that of control. According to Kaplan Meir’s curve, median survival of tumor bearing mice treated with PEITC was prolonged by 20.5%. Furthermore as compared to controls, we observed reduced HER2, EGFR and VEGF expression in the brain sections of PEITC treated mice. To the best of our knowledge, our study for the first time demonstrates the anti-metastatic effects of PEITC in vivo in a novel breast tumor metastasis model and provides the rationale for further clinical investigation.
Ovarian cancer is the leading cause of cancer related deaths in women. Genetic alterations including overexpression of EGFR play a crucial role in ovarian carcinogenesis. Here we evaluated the effect of phenethyl isothiocyanate (PEITC) in ovarian tumor cells in vitro and in vivo. Oral administration of 12 µmol PEITC resulted in drastically suppressing ovarian tumor growth in a preclinical mouse model. Our in vitro studies demonstrated that PEITC suppress the growth of SKOV-3, OVCAR-3 and TOV-21G human ovarian cancer cells by inducing apoptosis in a concentration-dependent manner. Growth inhibitory effects of PEITC were mediated by inhibition of EGFR and AKT, which are known to be overexpressed in ovarian tumors. PEITC treatment caused significant down regulation of constitutive protein levels as well as phosphorylation of EGFR at Tyr1068 in various ovarian cancer cells. In addition, PEITC treatment drastically reduced the phosphorylation of AKT which is downstream to EGFR and disrupted mTOR signaling. PEITC treatment also inhibited the kinase activity of AKT as observed by the down regulation of p-GSK in OVCAR-3 and TOV-21G cells. AKT overexpression or TGF treatment blocked PEITC induced apoptosis in ovarian cancer cells. These results suggest that PEITC targets EGFR/AKT pathway in our model. In conclusion, our study suggests that PEITC could be used alone or in combination with other therapeutic agents to treat ovarian cancer.
The title compound, C8H17O2PS2, displays a distorted tetrahedral geometry around the P atom. The P atom is part of a six-membered ring with an isopropyl group in the equatorial position. The molecules are linked by S—H⋯S hydrogen bonds in the crystal packing.
CpG-ODNs activate various immune cell subsets and induce the production of numerous cytokines. To determine whether a CpG-ODN-activated innate immune system, without the adaptive immune system, was capable of protecting against cancer cell growth, NOD/SCID mice, which do not have T or B cell function but have a functional innate immune system, were used as a model system. NOD/SCID mice were injected subcutaneously with human prostate cancer cells followed by subcutaneous injection of incremental doses of CpG-ODNs. CpG-ODNs displayed a dose-related antitumoral effect leading to the prevention of tumor growth. These results indicate that ODNs are capable of activating the innate immune system and destroying human cancer cells in the absence of the adaptive immune system.
CpG-ODNs; innate immune system; cancer; NOD-SCID mice
HER2 is an oncogene, expression of which leads to poor prognosis in 30% of breast cancer patients. Although trastuzumab is apparently an effective therapy against HER2-positive tumors, its systemic toxicity and resistance in the majority of patients restricts its applicability. In this study we evaluated the effects of phenethyl isothiocyanate (PEITC) in HER2-positive breast cancer cells.
MDA-MB-231 and MCF-7 breast cancer cells stably transfected with HER2 (high HER2 (HH)) were used in this study. The effect of PEITC was evaluated using cytotoxicity and apoptosis assay in these syngeneic cells. Western blotting was used to delineate HER2 signaling. SCID/NOD mice were implanted with MDA-MB-231 (HH) xenografts.
Our results show that treatment of MDA-MB-231 and MCF-7 cells with varying concentrations of PEITC for 24 h extensively reduced the survival of the cells with a 50% inhibitory concentration (IC50) of 8 μM in MDA-MB-231 and 14 μM in MCF-7 cells. PEITC treatment substantially decreased the expression of HER2, epidermal growth factor receptor (EGFR) and phosphorylation of signal transducer and activator of transcription 3 (STAT3) at Tyr-705. The expression of BCL-2-associated × (BAX) and BIM proteins were increased, whereas the levels of B cell lymphoma-extra large (BCL-XL) and X-linked inhibitor of apoptosis protein (XIAP) were significantly decreased in both the cell lines in response to PEITC treatment. Substantial cleavage of caspase 3 and poly-ADP ribose polymerase (PARP) were associated with PEITC-mediated apoptosis in MDA-MB-231 and MCF-7 cells. Notably, transient silencing of HER2 decreased and overexpressing HER2 increased the effects of PEITC. Furthermore, reactive oxygen species (ROS) generation, mitochondrial depolarization and apoptosis by PEITC treatment were much higher in breast cancer cells expressing higher levels of HER2 (HH) as compared to parent cell lines. The IC50 of PEITC following 24 h of treatment was reduced remarkably to 5 μM in MDA-MB-231 (HH) and 4 μM in MCF-7 (HH) cells, stably overexpressing HER2. Oral administration of 12 μM PEITC significantly suppressed the growth of breast tumor xenografts in SCID/NOD mice. In agreement with our in vitro results, tumors from PEITC-treated mice demonstrated reduced HER2, EGFR and STAT3 expression and increased apoptosis as revealed by cleavage of caspase 3 and PARP. In addition our results show that PEITC can enhance the efficacy of doxorubicin.
Our results show a unique specificity of PEITC in inducing apoptosis in HER2-expressing tumor cells in vitro and in vivo and enhancing the effects of doxorubicin. This unique specificity of PEITC offers promise to a subset of breast cancer patients overexpressing HER2.
apoptosis; doxorubicin; EGFR; ERBB2/HER2; in vivo; mitochondria; STAT3
Signal transducer and activator of transcription 3 (STAT3) is activated in majority of ovarian tumors and confers resistance to cisplatin treatment in patients with ovarian cancer. We have reported previously that diindolylmethane (DIM) inhibits the growth of ovarian cancer cells. However, to date the exact mechanism by which DIM induces growth suppressive effects has not been clear. In this report the mode of action of DIM is investigated.
Six human ovarian cancer cell lines and an ovarian tumor xenograft animal model were used to study the effect of diindolylmethane alone or in combination with cisplatin.
Diindolylmethane treatment induced apoptosis in all six ovarian cancer cell lines. Phosphorylation of STAT3 at Tyr-705 and Ser-727 was reduced by DIM in a concentration-dependent manner. In addition, diindolylmethane treatment inhibited nuclear translocation, DNA binding, and transcriptional activity of STAT3. Interleukin (IL)-6-induced phosphorylation of STAT3 at Tyr-705 was significantly blocked by DIM. Overexpression of STAT3 by gene transfection blocked DIM-induced apoptosis. In addition, DIM treatment reduced the levels of IL-6 in ovarian cancer cells and in the tumors. DIM treatment also inhibited cell invasion and angiogenesis by suppressing hypoxia-inducible factor 1α (HIF-1α) and vascular epithelial growth factor (VEGF). Importantly, diindolylmethane treatment potentiated the effects of cisplatin in SKOV-3 cells by targeting STAT3. Oral administration of 3 mg diindolylmethane per day and subsequent administration of cisplatin substantially inhibited in vivo tumor growth. Western blotting analysis of tumor lysates indicated increased apoptosis and reduced STAT3 activation.
These findings provide a rationale for further clinical investigation of DIM alone or in combination for chemoprevention and/or chemotherapy of ovarian cancer.
apoptosis; angiogenesis; cisplatin; diindolylmethane; STAT3