Repurposing Metabolic Inhibitors in the Treatment of Colon Adenocarcinoma Patient-Derived Models.

The effect of agonists on AMP-activated protein kinase (AMPK), mainly metformin and phenformin, has been appreciated in the treatment of multiple types of tumors. Specifically, the antitumor activity of phenformin has been demonstrated in melanomas containing the v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) activating mutation. In this report, we elucidated the synergistic antitumor effects of biguanides with metabolism inhibitors on colon tumors. Phenformin with 2-deoxy-D-glucose (2DG) inhibited tumor cell growth in cancer cell lines, including HT29 cells harboring BRAF- and p53-mutations. Biochemical analyses showed that two chemotherapeutics exerted cooperative effects to reduce tumor growth through cell cycle arrest, apoptosis, and autophagy. The drugs demonstrated activity against phosphorylated ERK and the gain-of-function p53 mutant protein. To demonstrate tumor regressive effects in vivo, we established patient-derived models, including xenograft (PDX) and organoids (PDO). Co-treatment of biguanides with chemotherapeutics efficiently reduced the growth of patient-derived colon models in comparison to treatment with a single agent. These results strongly suggest that significant therapeutic advantages would be achieved by combining AMPK activators such as phenformin and cancer metabolic inhibitors such as 2DG.

Evaluation of the Effect of Perfluorohexane Sulfonate on the Proliferation of Human Liver Cells.

Perfluorohexane sulfonate (PFHxS) is a widely detected replacement for legacy long-chain perfluoroalkyl substances (PFAS) in the environment and human blood samples. Its potential toxicity led to its recent classification as a globally regulated persistent organic pollutant. Although animal studies have shown a positive association between PFHxS levels and hepatic steatosis and hepatocellular hypertrophy, the link with liver toxicity, including end-stage liver cancer, remains inconclusive. In this study, we examined the effects of PFHxS on the proliferation of Hep3B (human hepatocellular carcinoma) and SK-Hep1 (human liver sinusoidal endothelial cells). Cells were exposed to different PFHxS concentrations for 24-48 h to assess viability and 12-14 days to measure colony formation. The viability of both cell lines increased at PFHxS concentrations <200 µM, decreased at >400 µM, and was highest at 50 µM. Colony formation increased at <300 µM and decreased at 500 µM PFHxS. Consistent with the effect on cell proliferation, PFHxS increased the expression of proliferating cell nuclear antigen (PCNA) and cell-cycle molecules (CDK2, CDK4, cyclin E, and cyclin D1). In summary, PFHxS exhibited a biphasic effect on liver cell proliferation, promoting survival and proliferation at lower concentrations and being cytotoxic at higher concentrations. This suggests that PFHxS, especially at lower concentrations, might be associated with HCC development and progression.

Acrylamide and its metabolite induce neurotoxicity via modulation of protein kinase C and AMP-activated protein kinase pathways.

Acrylamide is known as a neurotoxicant found in commonly consumed food as well as in human body. However, the underlying mechanisms involved in neurotoxicity by acrylamide and its metabolite, glycidamide remain largely unknown. In this study, we have examined the interplay between CYP2E1, AMPK, ERK and PKC in acrylamide-induced neurotoxicity associated with autophagy in PC12 cells. Acrylamide-induced cell death was mediated by CYP2E1 expression and the activation of ERK, PKC-ɑ and PKC-δ, whereas AMPK knockdown exacerbated the acrylamide-induced neurotoxic effects. PKC-ɑ, but not PKC-δ, plays an upstream regulator of ERK and AMPK. Moreover, AMPK activation suppressed ERK, and CYP2E1 and AMPK bilaterally inhibit each other. Furthermore, acrylamide increased autophagy with impaired autophagic flux, evidenced by the increased beclin-1, LC3-II and p62 protein. Acrylamide-induced neuronal death was ameliorated by 3-methyladenine, an autophagy inhibitor, whereas neuronal death was exacerbated by chloroquine, a lysosomal inhibitor. Interestingly, PKC-δ siRNA, but not PKC-ɑ siRNA, dramatically reduced acrylamide-induced beclin-1 and LC3-II levels, whereas AMPK siRNA further increased beclin-1, LC3-II and p62 protein levels. Glycidamide, a major metabolite, mimicked acrylamide only with a higher potency. Taken together, acrylamide- and glycidamide-induced neurotoxicity may involve cytotoxic autophagy, which is mediated by interplay between PKCs and AMPK pathways.

Mistletoe Extract Targets the STAT3-FOXM1 Pathway to Induce Apoptosis and Inhibits Metastasis in Breast Cancer Cells.

Mistletoe extracts (Viscum album L.) have been widely used as complementary and alternative medicines for the treatment of cancer, and their cytotoxic effects have been reported on various types of cancer. However, the molecular targets of mistletoe extracts have not been well studied. Herein, we investigated molecules associated with the in vitro and in vivo anticancer effects of mistletoe extract using 4T1 murine breast cancer cells. Mistletoe extract induced apoptosis and inhibited the signal transducer and activator of transcription3 (STAT3) phosphorylation. This inhibition was accompanied by the downregulations of forkhead box M1 (FOXM1) and the DNA repair proteins, RAD51 and survivin. Mistletoe extract simultaneously increased the expression of the DNA damage marker proteins, phosphorylated H2A histone family member X (H2A.X), and phosphorylated p38. Furthermore, mistletoe extract effectively suppressed tumor growth in 4T1 tumor-bearing BALB/c mice. In addition to tumor growth inhibition, mistletoe extract inhibited lung metastasis in the tumor-bearing mice and cell invasiveness by downregulating the expressions of matrix metalloproteinases (MMPs), urokinase-type plasminogen activator (uPA), uPA receptor, and markers of epithelial-mesenchymal transition (snail and fibronectin). Taken together, our results suggest that mistletoe extract targets the STAT3-FOXM1 pathway for its cytotoxic effects, and that mistletoe extracts might be useful for the treatment of patients with cancers highly expressing the STAT3-FOXM1 pathway.

Lotus (Nelumbo nucifera) seedpod extract inhibits cell proliferation and induces apoptosis in non-small cell lung cancer cells via downregulation of Axl.

Non-small-cell lung cancer (NSCLC) is the most frequent cause of cancer-related death. In this study, we found the anticancer activity of lotus seedpod extract (LSPE) in NSCLC cells, since LSPE treatment inhibited cell proliferation of A549 and H460 cells in a dose-dependent manner and the clonogenic activities of LSPE-treated cells were also reduced. In LSPE-treated cells, the cleavage of poly (ADP-ribose) polymerase (PARP) and phosphorylation of H2X, were also observed, indicating the pro-apoptotic effect of LSPE. Next, we found that LPSE treatment diminished the levels of protein and mRNA of Axl, a receptor tyrosine kinase (RTK) that transduces critical signals for cell proliferation and inhibition of apoptosis. The promoter activity of Axl was found to be dose-dependently decreased in response to LSPE treatment, implying that LSPE inhibited Axl gene expression at transcriptional level. In addition, Axl overexpression was found to decrease the effects of LSPE on inhibition of cell proliferation and colony formation as well as induction of PARP cleavage and phosphorylation of H2AX, while the same activities of LPSE were increased by knockdown of Axl gene expression, indicating that the antiproliferative and pro-apoptotic effect of LSPE is inversely proportional to the protein level of Axl. Taken together, we found that the LSPE suppressed cell proliferation and induced apoptosis of NSCLC cells, which is attenuated or augmented by overexpression or RNA interference of Axl expression, respectively. Our data suggest that Axl is a novel therapeutic target of LSPE to inhibit cell proliferation and promote apoptosis in NSCLC cells. PRACTICAL APPLICATIONS: In this study, lotus seedpod extract (LSPE) was found to have the cytotoxic and apoptosis-inducing potentials in non-small-cell lung cancer (NSCLC) cells. LSPE downregulated the Axl expression at transcriptional level and the effects of LSPE on cell proliferation as well as apoptosis were affected by Axl protein level. Therefore, the inference of Axl-mediated intracellular signals by LSPE must be a novel approach to control NSCLC. Since our data imply that LSPE contains bioactive compounds targeting Axl, further studies to elucidate these compounds might discover a potent therapeutic agent.

Bufalin down-regulates Axl expression to inhibit cell proliferation and induce apoptosis in non-small-cell lung cancer cells.

Axl, a member of the TAM (Tyro3, AXL, Mer) receptor tyrosine kinase family, plays critical roles in cell growth, proliferation, apoptosis, and migration. In the present study, we demonstrated that the anti-cancer activity of bufalin, a major bioactive component of the Chinese traditional medicine Chan Su, is mediated by the down-regulation of Axl in non-small-cell lung cancer (NSCLC) cells. We observed the inhibitory effect of bufalin on the proliferation of A549 and H460 NSCLC cells and the clonogenicity of these cells was reduced by bufalin treatment in a dose-dependent manner. Next, we found that the protein level of Axl was decreased in proportion to the concentration of bufalin in both A549 and H460 cells. Moreover, the promoter activity of the Axl gene was decreased by bufalin in a dose- and time-dependent manner, indicating that bufalin down-regulates Axl gene expression at the transcriptional level. We further examined if the anti-proliferative property of bufalin is influenced by Axl at the protein level. Axl overexpression attenuated the effect of bufalin in inhibiting cell proliferation and colony formation and inducing apoptosis in H460 cells, while knockdown of Axl gene expression induced the opposite effect. Taken together, our data indicate that the anti-proliferative and pro-apoptotic effects of bufalin were associated with the protein level of Axl, suggesting that Axl is a potent therapeutic target of bufalin in suppressing proliferation and inducing apoptosis in NSCLC cells.

Taxotere Induces Dephosphorylation of MET in Patient-derived Tumor Models.

BACKGROUND/AIM: Although molecular targeting therapy is an attractive treatment for cancer, resistance eventually develops in most cases. Here, we evaluated chemotherapeutic efficacy on non-small cell lung cancer (NSCLC) with acquired resistance to epidermal growth factor receptor inhibitors mechanistically. MATERIALS AND METHODS: Antitumor effects of taxotere were evaluated using multiple models, including xenograft, and patient-derived models developed from adenocarcinoma cancer patients. Protein expressions were analyzed after drug treatment. RESULTS: Taxotere inhibited tumor growth of NSCLC cells harboring drug resistance, and reduced the expression of phosphorylated MET proto-oncogene, receptor tyrosine kinase (MET). A tumor-inhibitory effect of taxotere was also demonstrated in vivo in xenografts in mice, patient-derived primary lung tumor cells and patient-derived xenograft with concomitant repression of phosphorylated MET expression. Chemotherapeutic and MET-targeting drug exhibited a synergistic cell growth-inhibitory effect. CONCLUSION: These results suggest that the anticancer drug taxane may be an adjuvant for lung tumors exhibiting enhanced signaling of MET networks.

Axl is a novel target of celastrol that inhibits cell proliferation and migration, and increases the cytotoxicity of gefitinib in EGFR mutant non­small cell lung cancer cells.

Gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR­TKI) is an excellent therapeutic agent to treat EGFR mutation­positive non­small cell lung cancer (NSCLC). However, the initial response decreases as chemoresistance develops. In the present study, gefitinib­resistant EGFR mutant NSCLC PC­9/GR cells were established to examine the characteristics and mechanisms associated with chemoresistance. Axl expression in PC­9/GR cells was transcriptionally upregulated, since Axl protein and mRNA expression levels were identified to be increased according to western blot analysis and reverse transcription polymerase chain reaction results. The inhibitory effect of celastrol on Axl protein expression level, cell viability and clonogenicity were identified in parental and gefitinib­resistant PC­9 cells. In addition, treatment of PC­9/GR cells with celastrol and gefitinib in combination was demonstrated to synergistically suppress Axl protein expression level, cell proliferation and migration. Taken together, upregulation of Axl expression seems to be associated with chemoresistance of PC­9/GR cells. Furthermore, celastrol targets Axl to exert its anticancer effects in order to increase the susceptibility of PC­9/GR cells to gefitinib and overcome chemoresistance.

Metformin selectively targets 4T1 tumorspheres and enhances the antitumor effects of doxorubicin by downregulating the AKT and STAT3 signaling pathways.

Recent studies have reported that metformin (Met), the first-line medication for the treatment of type 2 diabetes, exhibited anticancer and chemoprotective effects in diverse cancer cells. In this study, we investigated the effects of Met on the drug-resistance of 4T1 murine breast cancer tumorspheres (TS) and the mechanism responsible for its drug-resistance. 4T1 TS exhibited accumulations of cells at the G0/G1 phase compared with cells in monolayer culture, which suggested the majority of cells in TS were quiescent. Furthermore, it was identified that activations of the signal transducer and activator of transcription 3 (STAT3) and protein kinase B (AKT) signaling pathways in 4T1 TS conferred drug-resistance to doxorubicin (Dox) and lapatinib (Lapa). However, Met selectively targeted TS rather than cells in monolayer culture and increased the cytotoxic effect of Dox on TS by inhibiting activations of the STAT3 and AKT signaling pathways. These observations suggested that inhibitions of STAT3 and AKT underlie the selective cytotoxic effects of Met on TS. In addition, Met exhibited synergistic antitumor effects with Dox on 4T1 tumor-bearing BALB/c mice. Our findings suggest that combinations of Met and cytotoxic anticancer drugs may offer an advantage for treating drug-resistant breast cancer.

Carvacrol Targets AXL to Inhibit Cell Proliferation and Migration in Non-small Cell Lung Cancer Cells.

BACKGROUND/AIM: AXL has been reported to be overexpressed and highly activated in various cancer types. In this study, we demonstrated the effect of carvacrol on cell proliferation and migration in non-small cell lung cancer (NSCLC) cells by impeding the expression and activation of AXL. MATERIALS AND METHODS: The levels of AXL protein, mRNA and promoter activity were evaluated by western blot, reverse transcription polymerase chain reaction (RT-PCR) and luciferase assay, respectively. AXL-overexpressing cells were established by ectopic expression of AXL cDNA. Cell viability, clonogenicity, and migration were measured in carvacrol-treated NSCLC cells. RESULTS: Carvacrol treatment of NSCLC cells caused down-regulation of AXL expression at the transcriptional level and also inhibited phosphorylation of AXL upon ligand stimulation. Carvacrol suppressed cell proliferation and migration and its inhibitory effect was attenuated in AXL-overexpressing NSCLC cells. CONCLUSION: Our data demonstrate that AXL is a crucial therapeutic target of carvacrol-induced inhibition of NSCLC cell proliferation and migration.

Mistletoe (Viscum album) extract targets Axl to suppress cell proliferation and overcome cisplatin- and erlotinib-resistance in non-small cell lung cancer cells.

BACKGROUND: Mistletoe extract of Visucm album extract (VAE) contains many biologically active components and has been reported to be not only a complementary and alternative medicine, but also a potent therapeutic agent for many types of cancer. PURPOSE: In this study, we examined the effect of VAE on expression and activation of Axl and scrutinized the involvement of Axl in the anti-cancer activity of VAE in parental and chemo-resistant non-small cell lung cancer (NSCLC) cells. METHODS: The levels of Axl protein and mRNA were determined by Western blot analysis and RT-PCR, respectively. Phosphorylation of Axl upon Gas6 stimulation was observed by Western blot analysis. For ectopic expression or gene silencing of Axl, the recombinant plasmid, pcDNA3-Axl, or specific siRNA targeting Axl were transfected into A549 and H460 cells using Lipofectamine 2000, respectively. The anti-cancer activity of mistletoe extract was examined against the parental cells and each of their cisplatin- or erlotinib-resistant cells using trypan blue exclusion assays and colony formation assay. RESULTS: The levels of Axl mRNA were also reduced by VAE treatment, implying the transcriptional downregulation of Axl expression by VAE. In addition, the phosphorylation of Axl protein upon its ligand, Gas6, stimulation was found to be abrogated by VAE. We next found cytotoxic effect of VAE on both the parental NSCLC cells and their variants which are resistant to cisplatin (A549/CisR and H460/CisR) or erlotinib (H460/ER and H1975/ER). Treatment of these cells with VAE caused a dose-dependent decrease of cell viability and clonogenicity. This anti-proliferative effect of VAE was attenuated in Axl-overexpressing cells, while it was augmented in cells transfected Axl specific siRNA. Next, we also found that in cisplatin-resistant cells and erlotinib-resistant cells, VAE treatment decreased Axl protein level, colonogenicity. The levels of several cell cycle regulator, p21 and apoptosis related protein, X-linked inhibitor of apoptosis, was found to be induced and reduced by VAE treatment, respectively. CONCLUSION: Taken together, our data provide that VAE targets Axl to suppress cell proliferation and to circumvent cisplatin- and erlotinib-resistance in NSCLC cells.

Development and validation of a subject-specific finite element model of the functional spinal unit to predict vertebral strength.

Finite element models of an isolated vertebral body cannot accurately predict compressive strength of the spinal column because, in life, compressive load is variably distributed across the vertebral body and neural arch. The purpose of this study was to develop and validate a patient-specific finite element model of a functional spinal unit, and then use the model to predict vertebral strength from medical images. A total of 16 cadaveric functional spinal units were scanned and then tested mechanically in bending and compression to generate a vertebral wedge fracture. Before testing, an image processing and finite element analysis framework (SpineVox-Pro), developed previously in MATLAB using ANSYS APDL, was used to generate a subject-specific finite element model with eight-node hexahedral elements. Transversely isotropic linear-elastic material properties were assigned to vertebrae, and simple homogeneous linear-elastic properties were assigned to the intervertebral disc. Forward bending loading conditions were applied to simulate manual handling. Results showed that vertebral strengths measured by experiment were positively correlated with strengths predicted by the functional spinal unit finite element model with von Mises or Drucker-Prager failure criteria ( R2 = 0.80-0.87), with areal bone mineral density measured by dual-energy X-ray absorptiometry ( R2 = 0.54) and with volumetric bone mineral density from quantitative computed tomography ( R2 = 0.79). Large-displacement non-linear analyses on all specimens did not improve predictions. We conclude that subject-specific finite element models of a functional spinal unit have potential to estimate the vertebral strength better than bone mineral density alone.

Anticancer effect of luteolin is mediated by downregulation of TAM receptor tyrosine kinases, but not interleukin-8, in non-small cell lung cancer cells.

TAM receptor tyrosine kinases (RTKs), Tyro3, Axl and MerTK, transduce diverse signals responsible for cell survival, growth, proliferation and anti-apoptosis. In the present study, we demonstrated the effect of luteolin, a flavonoid with antioxidant, anti-inflammatory and anticancer activities, on the expression and activation of TAM RTKs and the association with its cytotoxicity in non-small cell lung cancer (NSCLC) cells. We observed the cytotoxic effect of luteolin in parental A549 and H460 cells as well as in cisplatin-resistant A549/CisR and H460/CisR cells. Exposure of these cells to luteolin also resulted in a dose­dependent decrease in clonogenic ability. Next, luteolin was found to decrease the protein levels of all three TAM RTKs in the A549 and A549/CisR cells in a dose­dependent manner. In a similar manner, in H460 and H460/CisR cells, the protein levels of Axl and Tyro3 were decreased following luteolin treatment. In addition, Axl promoter activity was decreased by luteolin, indicating that luteolin suppresses Axl expression at the transcriptional level. We next found that luteolin abrogated Axl phosphorylation in response to growth arrest-specific 6 (Gas6), its ligand, implying the inhibitory effect of luteolin on Gas6-induced Axl activation. Ectopic expression of Axl was observed to attenuate the antiproliferative effect of luteolin, while knockdown of the Axl protein level using a gold nanoparticle-assisted gene delivery system increased its cytotoxicity. In contrast to the inhibitory effect of luteolin on the expression of TAM RTKs, interleukin-8 (IL-8) production was not decreased by luteolin in H460 and H460/CisR cells, while IL-8 production/cell was increased. Collectively, our data suggest that TAM RTKs, but not IL-8, are promising therapeutic targets of luteolin to abrogate cell proliferation and to overcome chemoresistance in NSCLC cells.

Suppression of the metastatic spread of breast cancer by DN10764 (AZD7762)-mediated inhibition of AXL signaling.

Breast cancer is the most common malignant disease occurring in women and represents a substantial proportion of the global cancer burden. In these patients, metastasis but not the primary tumor is the main cause of breast cancer-related deaths. Here, we report the novel finding that DN10764 (AZD7762, a selective inhibitor of checkpoint kinases 1 and 2) can suppress breast cancer metastasis. In breast cancer cells, DN10764 inhibited cell proliferation and GAS6-mediated AXL signaling, consequently resulting in suppressed migration and invasion. In addition, DN10764 induced caspase 3/7-mediated apoptosis in breast cancer cells and inhibited tube formation of human umbilical vein endothelial cells. Finally, DN10764 significantly suppressed the tumor growth and metastasis of breast cancer cells in in vivo metastasis models. Taken together, these data suggest that therapeutic strategies targeting AXL in combination with systemic therapies could improve responses to anti-cancer therapies and reduce breast cancer recurrence and metastases.

Suppression of gain-of-function mutant p53 with metabolic inhibitors reduces tumor growth in vivo.

Mutation of p53 occasionally results in a gain of function, which promotes tumor growth. We asked whether destabilizing the gain-of-function protein would kill tumor cells. Downregulation of the gene reduced cell proliferation in p53-mutant cells, but not in p53-null cells, indicating that the former depended on the mutant protein for survival. Moreover, phenformin and 2-deoxyglucose suppressed cell growth and simultaneously destabilized mutant p53. The AMPK pathway, MAPK pathway, chaperone proteins and ubiquitination all contributed to this process. Interestingly, phenformin and 2-deoxyglucose also reduced tumor growth in syngeneic mice harboring the p53 mutation. Thus, destabilizing mutant p53 protein in order to kill cells exhibiting "oncogene addiction" could be a promising strategy for combatting p53 mutant tumors.

Curcumin-induced downregulation of Axl receptor tyrosine kinase inhibits cell proliferation and circumvents chemoresistance in non-small lung cancer cells.

Lung cancer is still in the first place in terms of both incidence and mortality. In the present study, we demonstrated the effect of curcumin, a phytochemical of the plant Curcuma longa, on expression and activation of Axl receptor tyrosine kinase (RTK) which plays an important role in cell survival, proliferation and anti-apoptosis. Curcumin treatment of non-small cell lung cancer (NSCLC) A549 and H460 cells, was found to decrease Axl protein as well as mRNA levels in a dose- and time-dependent manner. Axl promoter activity was also reduced by curcumin, indicating that curcumin downregulates Axl expression at the transcriptional level. Moreover, Axl phosphorylation in response to binding of its ligand, Gas6, was abrogated by curcumin, suggesting the inhibitory effect of curcumin on Gas6-induced Axl activation. We next found cytotoxic effect of cucumin on both the parental A549 and H460 cells, and their variants which are resistant to cisplatin (A549/CisR and H460/CisR) and paclitaxel (A549/TR and H460/TR). Exposure of these cells to curcumin resulted in dose-dependent decline of cell viability and clonogenic ability. It is further observed that the anti-proliferative effect of curcumin on A549 cells overexpressing Axl protein was reduced, while that on H460 cells transfected Axl specific siRNA was augmented, confirming that curcumin inhibits cell proliferation via downregulation of Axl expression. In addition, curcumin was found to cause the induction of p21, a cyclin-dependent kinase inhibitor, and reduction of X-linked inhibitor of apoptosis protein (XIAP), an anti-apoptotic molecule, in parental H460 cells as well as chemoresistant cells, H460/CisR and H460/TR. Taken together, our data imply that Axl RTK is a novel target of curcumin through which it exerts anti-proliferative effect in both parental and chemoresistant NSCLC cells.

Metformin targets Axl and Tyro3 receptor tyrosine kinases to inhibit cell proliferation and overcome chemoresistance in ovarian cancer cells.

Metformin, the most frequently prescribed anti-diabetic drug, has recently been paid attention as a chemotherapeutic agent. In this study, we demonstrated that metformin decreased the viability of parental as well as cisplatin/taxol-resistant ovarian cancer cells. Its anti-proliferative effect was further demonstrated by dose­dependent reduction of the clonogenic ability of the metformin­treated cells. We next observed the effect of metformin on expression of Axl and Tyro3 receptor tyrosine kinases (RTKs) which belong to the TAM subfamily of RTKs transducing pro-survival and anti-apoptotic signals. Metformin treatment of ovarian cancer cells decreased both mRNA and protein levels of Axl and Tyro3 in a dose­dependent manner. Axl promoter activity was also inhibited by metformin, indicating that metformin suppresses Axl and Tyro3 expression at the transcriptional level. Metformin treatment was also found to augment its anti­proliferative effect in SKOV3 and taxol-resistant SKOV3/TR cells transfected with Axl and Tyro3 specific siRNAs, siAxl and siTyro3, respectively, suggesting that metformin might target Axl and Tyro3 RTKs to restrain cell proliferation. In parallel, the level of X-linked inhibitor of apoptosis protein (XIAP), an anti-apoptotic molecule, was reduced in the metformin-treated cells. Collectively, our data showed that metformin caused reduction of Axl and Tyro3 RTKs' expression, inactivation of downstream effectors, and decrease of anti-apoptotic protein level, forming a potent therapeutic strategy to facilitate its anticancer activity as well as to overcome chemoresistance in human ovarian cancer cells.

Overexpression of Tyro3 receptor tyrosine kinase leads to the acquisition of taxol resistance in ovarian cancer cells.

The majority of patients with ovarian cancer are diagnosed at the advanced stages (III/IV) and their 5-year-survival rate is relatively low. One of the major causes of the poor prognosis of ovarian cancer is the development of resistance to first-line chemotherapy, including platinum and taxol. Therefore, improvements in current understanding of chemoresistance is required for the successful treatment of ovarian cancer. In the present study, taxol-resistant ovarian cancer cells, SKOV3/TR, were established by exposing parental SKOV3 cells to increasing concentrations of taxol. . Briefly, cells were treated with 1.5 nM (for 4 weeks), 3 nM (for 4 weeks), 6 nM (for 5 weeks), 12 nM (for 5 weeks) and 24 nM taxol (for 8 weeks) over 6 months. The SKOV3/TR cells were found to be smaller in size and rounder in shape compared with their parental cells. Cell viability and colony formation assays demonstrated an increase in the population doubling time of the SKOV3/TR cells, indicating a reduction in the proliferative capacity of these cells. Reverse transcription-polymerase chain reaction and western blot analysis revealed that, among the TAM receptor tyrosine kinases (RTKs), the mRNA and protein expression levels of Tyro3 RTK were increased, while those of Axl and Mer RTK were decreased in the SKOV3/TR cells. In addition, restoration of the level of Tyro3 by transfecting Tyro3-specific small interfering RNA into the SKOV3/TR cells reduced the proliferative capacity of the cells, indicating that upregulation of the expression of Tyro3 in SKOV3/TR cells may promote survival in the presence of taxol, which eventually resulted in the acquisition of resistance upon taxol treatment. The present study subsequently found that, in the SKOV3/TR cells, the level of intracellular reactive oxygen species (ROS) was elevated, and antioxidant treatment with N-acetyl cysteine (NAC) exerted more profound antiproliferative effects compared with the parental cells. The western blot analysis demonstrated that treatment of the SKOV3/TR cells with NAC reduced the protein expression of Tyro3, and the inhibitory effect of NAC on the phosphorylation of Akt was increased, which may have had a positive effect on the proliferation of the SKOV3/TR cells. The levels of phosphorylation and protein expression of signal transducers and activators of transcription 3 (STAT3) were not affected by NAC treatment, indicating that the phosphorylation of Akt, but not expression or phosphorylation of STAT3, was associated with the increased intracellular ROS level in the SKOV3/TR cells. Taken together, the results of the present study demonstrated that the acquired taxol resistance of ovarian cancer cells was associated with ROS-dependent upregulation in the expression of Tyro3 RTK and the subsequent activation of Akt.

RGS2 suppresses breast cancer cell growth via a MCPIP1-dependent pathway.

Regulator of G protein signaling 2 (RGS2) is a member of a family of proteins that functions as a GTPase-activating protein (GAP) for Gα subunits. RGS2 mRNA expression is lower in breast cancerous tissues than in normal tissues. In addition, expression of RGS2 is also lower in MCF7 (cancerous breast cells) than in MCF10A (normal breast cells). Here we investigated whether RGS2 inhibits growth of breast cancer cells. RGS2 overexpression in MCF7 cells inhibited epidermal growth factor- or serum-induced proliferation. In HEK293T cells expressing RGS2, cell growth was also significantly suppressed (In addition, exogenous expression of RGS2 in HEK293T cells resulted in the significant suppression of cell growth). These results suggest that RGS2 may have a tumor suppressor function. MG-132 treatment of MCF7 cells increased endogenous or exogenous RGS2 levels, suggesting a post-transcriptional regulatory mechanism that controls RGS2 protein levels. RGS2 protein was degraded polyubiquitinated the K71 residue, but stabilized by deubiquitinase monocyte chemotactic protein-induced protein 1 (MCPIP1), and not affected by dominant negative mutant (C157A) of MCPIP1. Gene expression profiling study showed that overexpression of RGS2 decreased levels of testis specific Y encoded like protein 5 (TSPYL5), which plays a causal role in breast oncogenesis. TSPYL5 protein expression was low in MCF10A and high in MCF7 cells, showing the opposite aspect to RGS2 expression. Additionally, RGS2 or MCPIP1 overexpression in MCF7 cells decreased TSPYL5 protein level, indicating that RGS2 stabilized by MCPIP1 have diminished TSPYL5 protein levels, thereby exerting an inhibitory effect of breast cancer cell growth.

Inhibition of IL-6/STAT3 axis and targeting Axl and Tyro3 receptor tyrosine kinases by apigenin circumvent taxol resistance in ovarian cancer cells.

Ovarian cancer is the number one cause of death from gynaecological malignancy. Platinum-based and taxol-based chemotherapy has been used as a standard therapy, but intrinsic and acquired resistance to chemotherapy is a major obstacle to treat the disease. In the present study, we found that in the chemoresistant ovarian cancer SKOV3/TR cells, interleukin-6 (IL-6), IL-6 receptor and signal transducers and activators of transcription 3 (STAT3) expression as well as STAT3 phosphorylation were upregulated compared to those in parental cells. Silencing of IL-6 using IL-6 siRNA was found to suppress IL-6 production, STAT3 and phosphoSTAT3 levels, which eventually reduced proliferation and clonogenicity of taxol-resistant SKOV3/TR cells. In addition, stattic, a STAT3 inhibitor, was found to result in decrease of cell viability and clonogenicity of these cells, indicating that the elevated IL-6 and STAT3, phosphoSTAT3 levels are associated with the development of taxol resistance. Next, we found anti-proliferative effect of apigenin on both SKOV3 and SKOV3/TR cells. RT-PCR and western blot results showed that apigenin significantly reduced the expression of Axl and Tyro3 receptor tyrosine kinases (RTKs) at mRNA and protein level, which account for its cytotoxic activity. We further found that apigenin decreased Akt phosphorylation and the level of B-cell lymphoma-extra large (Bcl-xl or BCL2-like 1 isoform 1), an inhibitor of apoptosis. On the contrary to these results, apigenin had no effect on IL-6 production, STAT3 and phosphoSTAT3 protein levels, suggesting that apigenin exerts its anti-proliferative activity via downregulation of Axl and Tyro3 expression, Akt phosphorylation and Bcl-xl expression, but not modulation of IL-6/STAT3 axis. Taken together, our data suggest that inhibition of IL-6/STAT3 signaling pathway and downregulation of Axl and Tyro3 RTKs expression might be a therapeutic strategy to overcome taxol resistance in ovarian cancer cells.