Jeng Fong Chiou

Trifluoperazine, an Antipsychotic Agent, Inhibits Cancer Stem Cell Growth and Overcomes Drug Resistance of Lung Cancer

RATIONALE Cancer stem cell (CSC) theory has drawn much attention, with evidence supporting the contribution of stem cells to tumor initiation, relapse, and therapy resistance. OBJECTIVES To screen drugs that target CSCs to improve the current treatment outcome and overcome drug resistance in patients with lung cancer. METHODS We used publicly available embryonic stem cell and CSC-associated gene signatures to query the Connectivity Map for potential drugs that can, at least in part, reverse the gene expression profile of CSCs. High scores were noted for several phenothiazine-like antipsychotic drugs, including trifluoperazine. We then treated lung CSCs with different EGFR mutation status with trifluoperazine to examine its anti-CSC properties. Lung CSCs resistant to epidermal growth factor receptor-tyrosine kinase inhibitor or cisplatin were treated with trifluoperazine plus gefitinib or trifluoperazine plus cisplatin. Animal models were used for in vivo validation of the anti-CSC effect and synergistic effect of trifluoperazine with gefitinib. MEASUREMENTS AND MAIN RESULTS We demonstrated that trifluoperazine inhibited CSC tumor spheroid formation and down-regulated the expression of CSC markers (CD44/CD133). Trifluoperazine inhibited Wnt/β-catenin signaling in gefitinib-resistant lung cancer spheroids. The combination of trifluoperazine with either gefitinib or cisplatin overcame drug resistance in lung CSCs. Trifluoperazine inhibited the tumor growth and enhanced the inhibitory activity of gefitinib in lung cancer metastatic and orthotopic CSC animal models. CONCLUSIONS Using in silico drug screening by Connectivity Map followed by empirical validations, we repurposed an existing phenothiazine-like antipsychotic drug, trifluoperazine, as a potential anti-CSC agent that could overcome epidermal growth factor receptor-tyrosine kinase inhibitor and chemotherapy resistance.

Pterostilbene, a bioactive component of blueberries, suppresses the generation of breast cancer stem cells within tumor microenvironment and metastasis via modulating NF‐κB/microRNA 448 circuit

SCOPE Tumor-associated macrophages (TAMs) have been shown to promote metastasis and malignancy. Pterostilbene, a natural stilbene isolated from blueberries, has been suggested for anti-cancer effects. Here, we explored the potential cancer stem cells (CSCs)/TAM modulating effects of pterostilbene in breast cancer. METHODS AND RESULTS Using flowcytometric and Boyden chamber assay, we showed MCF7 and MDA-MB-231 cells cocultured with M2 TAMs exhibited increased percentage of CD44(+) /CD24(-) CSC population and migratory/invasive abilities. RT-PCR results showed that CD44(+) /CD24(-) cells expressed an increased level of HIF-1α, β-catenin, Twist1, and NF-κB and enhanced tumor sphere forming ability. Additionally, pterostilbene treatment dose dependently overcame M2 TAM-induced enrichment of CSCs and metastatic potential of breast cancer cells. Mechanistically, pterostilbene suppressed NFκB, Twist1, vimentin, and increased E-cadherin expression. Using siRNA technique, we demonstrated that pterostilbene-mediated NFκB downregulation was correlated to an increased amount of microRNA 448. Finally, pterostilbene-mediated suppression in tumorigenesis and metastasis was validated by noninvasive bioluminescence in mice bearing M2 TAM cocultured MDA-MB-231 tumor. CONCLUSION Pterostilbene effectively suppresses the generation of CSCs and metastatic potential under the influence of M2 TAMs via modulating EMT associated signaling pathways, specifically NF-κB/miR488 circuit. Thus, pterostilbene could be an ideal anti-CSC agent in clinical settings.

DSG3 Facilitates Cancer Cell Growth and Invasion through the DSG3-Plakoglobin-TCF/LEF-Myc/Cyclin D1/MMP Signaling Pathway

Desmoglein 3 (DSG3) is a component of the desmosome, which confers strong cell-cell adhesion. Previously, an oncogenic function of DSG3 has been found in head neck cancer (HNC). Here, we investigated how this molecule contributes to the malignant phenotype. Because DSG3 is associated with plakoglobin, we examined whether these phenotypic alterations were mediated through the plakoglobin molecule. Immunoprecipitation and immunofluorescence staining revealed that DSG3 silencing disrupted its interaction with plakoglobin and induced plakoglobin translocation from the cytoplasm to the nucleus. Knockdown of DSG3 significantly increased the interaction of plakoglobin with the transcriptional factor TCF and suppressed the TCF/LEF transcriptional activity. These effects further conferred to reduced expression of the TCF/LEF downstream target genes, including c-myc, cyclin D1, and MMP-7. Functional analyses showed that DSG3 silencing reduced cell growth and arrested cells at G0/G1 phase. Besides, cell migration and invasion abilities were also decreased. These cellular results were confirmed using tumor xenografts in mice, as DSG3 silencing led to the suppressed tumor growth, plakoglobin translocation and reduced expression of TCF/LEF target genes in tumors. Therefore, our study shows that the desmosomal protein DSG3 additionally functions to regulate malignant phenotypes via nuclear signaling. In conclusion, we found that DSG3 functions as an oncogene and facilitates cancer growth and invasion in HNC cells through the DSG3-plakoglobin-TCF/LEF pathway.

The effect of diminished osteogenic signals on reduced osteoporosis recovery in aged mice and the potential therapeutic use of adipose-derived stem cells

Adipose-derived stem cells (ADSCs) have been shown to be pluoripotent and explored for their usage in tissue engineering. Previously, we have established a cell-based approach comprised of platelet-enriched plasma and osteo-progenitor cells for treating osteoporosis in an ovariectomized-senescence-accelerated mice (OVX-SAMP8) model. In the present study, we intend to explore the feasibility of using ADSCs as a cell-based therapeutic approach for treating osteoporosis, and to examine the effects of aging on the pluoripotency of ADSCs and the efficiency of bone formation both in vitro and in vivo. Flow cytometry was used to characterize ADSCs isolated from young and aged female SAMP8 mice and showed that the highly positive expression of surface markers such as CD44 and CD105 and negative for CD34 and CD45. Therefore, to compare the aging effects on the growth kinetics and differentiation potential of young and aged ADSCs, we found that there was a significant decline in both the proliferation rate (approximately 13.3%) and osteo-differentiation potential in aged ADSC. Subsequently, young and aged ADSCs were transplanted into the bone marrow of osteoporotic mice (OVX-SAMP8) to evaluate their bone formation ability. ADSC transplants were shown effective in restoring bone mineral density in the right/left knees, femurs and spine, 4 months post-transplantation; mice which received young ADSC transplants showed significantly higher bone regeneration (an average of 24.3% of improved BMD) over those received aged ADSCs. In conclusion, these findings showed that aging impedes osteoporosis-ameliorating potential of ADSC by diminishing osteogenic signal, and that ADSC could be used as a potential cell-based therapy for osteoporosis.

Increased cellular apoptosis susceptibility (CSE1L/CAS) protein expression promotes protrusion extension and enhances migration of MCF-7 breast cancer cells

Microtubules are part of cell structures that play a role in regulating the migration of cancer cells. The cellular apoptosis susceptibility (CSE1L/CAS) protein is a microtubule-associated protein that is highly expressed in cancer. We report here that CSE1L regulates the association of α-tubulin with β-tubulin and promotes the migration of MCF-7 breast cancer cells. CSE1L was associated with α-tubulin and β-tubulin in GST (glutathione S-transferase) pull-down and immunoprecipitation assays. CSE1L-GFP (green fluorescence protein) fusion protein experiments showed that the N-terminal of CSE1L interacted with microtubules. Increased CSE1L expression resulted in decreased tyrosine phosphorylation of α-tubulin and β-tubulin, increased α-tubulin and β-tubulin association, and enhanced assembly of microtubules. Cell protrusions or pseudopodia are temporary extensions of the plasma membrane and are implicated in cancer cell migration and invasion. Increased CSE1L expression increased the extension of MCF-7 cell protrusions. In vitro migration assay showed that enhanced CSE1L expression increased the migration of MCF-7 cells. Our results indicate that CSE1L plays a role in regulating the extension of cell protrusions and promotes the migration of cancer cells.

CSE1L, a Novel Microvesicle Membrane Protein, Mediates Ras-Triggered Microvesicle Generation and Metastasis of Tumor Cells

Tumor-derived microvesicles are rich in metastasis-related proteases and play a role in the interactions between tumor cells and tumor microenvironment in tumor metastasis. Because shed microvesicles may remain in the extracellular environment around tumor cells, the microvesicle membrane protein may be the potential target for cancer therapy. Here we report that chromosome segregation 1-like (CSE1L) protein is a microvesicle membrane protein and is a potential target for cancer therapy. v-H-Ras expression induced extracellular signal-regulated kinase (ERK)-dependent CSE1L phosphorylation and microvesicle biogenesis in various cancer cells. CSE1L overexpression also triggered microvesicle generation, and CSE1L knockdown diminished v-H-Ras-induced microvesicle generation, matrix metalloproteinase (MMP)-2 and MMP-9 secretion and metastasis of B16F10 melanoma cells. CSE1L was preferentially accumulated in microvesicles and was located in the microvesicle membrane. Furthermore, anti-CSE1L antibody-conjugated quantum dots could target tumors in animal models. Our findings highlight a novel role of Ras-ERK signaling in tumor progression and suggest that CSE1L may be involved in the “early” and “late” metastasis of tumor cells in tumorigenesis. Furthermore, the novel microvesicle membrane protein, CSE1L, may have clinical utility in cancer diagnosis and targeted cancer therapy.

Transplantation of Embryonic Fibroblasts Treated with Platelet-Rich Plasma Induces Osteogenesis in SAMP8 Mice Monitored by Molecular Imaging

The aim of this study was to develop a cell-based bone-regeneration approach evaluated by molecular imaging and immunohistochemistry. Methods: Genetically modified NIH3T3 embryonic fibroblasts carrying enhanced green fluorescent protein (NIH3T3-G) were predifferentiated into osteoblastlike cells using platelet-rich plasma (PRP) medium, followed by intraosseous transplantation into ovariectomized senescence-accelerated mouse prone substrain 8 (OVX-SAMP8 mice). Results: PRP-conditioned NIH3T3-G (PRP/NIH3T3-G) engraftment prevented the development of osteoporosis. Molecular imaging and immunohistochemistry demonstrated the migration of NIH3T3-G cells from the implantation site throughout the skeleton. In situ analyses revealed coexpression of osteopontin and green fluorescent protein in the newly formed bone tissue, demonstrating that the transplant restored the bone trabecular architecture and mineral density in treated OVX-SAMP8 mice. Interestingly, the life span of OVX-SAMP8 mice receiving PRP/NIH3T3-G transplantation was significantly prolonged and similar to that of the congenic senescence-resistant strain of mice. Conclusion: This unique and yet simple approach could potentially be applied to the treatment of senile postmenopausal osteoporosis and perhaps inborn genetic syndromes associated with accelerated aging, such as Hutchinson–Gilford progeria syndrome, and for the prolongation of life expectancy in general.

BlueBerry Isolate, Pterostilbene, Functions as a Potential Anticancer Stem Cell Agent in Suppressing Irradiation-Mediated Enrichment of Hepatoma Stem Cells.

For many malignancies, radiation therapy remains the second option only to surgery in terms of its curative potential. However, radiation-induced tumor cell death is limited by a number of factors, including the adverse response of the tumor microenvironment to the treatment and either intrinsic or acquired mechanisms of evasive resistance, and the existence of cancer stem cells (CSCs). In this study, we demonstrated that using different doses of irradiation led to the enrichment of CD133+ Mahlavu cells using flow cytometric method. Subsequently, CD133+ Mahlavu cells enriched by irradiation were characterized for their stemness gene expression, self-renewal, migration/invasion abilities, and radiation resistance. Having established irradiation-enriched CD133+ Mahlavu cells with CSC properties, we evaluated a phytochemical, pterostilbene (PT), found abundantly in blueberries, against irradiation-enriched CSCs. It was shown that PT treatment dose-dependently reduced the enrichment of CD133+ Mahlavu cells upon irradiation; PT treatment also prevented tumor sphere formation, reduced stemness gene expression, and suppressed invasion and migration abilities as well as increasing apoptosis of CD133+ Mahlavu CSCs. Based on our experimental data, pterostilbene could be used to prevent the enrichment of CD133+ hepatoma CSCs and should be considered for future clinical testing as a combined agent for HCC patients.

Normoxically Overexpressed Hypoxia Inducible Factor 1-Alpha is Involved in Arsenic Trioxide Resistance Acquisition in Hepatocellular Carcinoma

BackgroundThe aim of this study was to examine the underlying signaling mechanisms of arsenic trioxide (ATO)-mediated anticancer effects and the responsible biomarker(s) for the acquired resistance in human heptatocellular carcinoma (HCC).Materials and MethodsThe therapeutic effects of ATO were examined using 2 characteristically distinct HCC cell lines, Hep-J5 (overexpressing HIF-1α/GRP78) and SK-Hep-1 (the matched control). ATO-mediated proliferation inhibition, oxidative stress, and apoptosis were analyzed using flowcytometric analysis and western blotting. The role of HIF-1α and GRP78 in HCC resistance to ATO treatment was determined using RNA silencing and inhibitor approaches.ResultsSK-Hep-1 cells, lacking both HIF-1α and GRP78 expressions were responsive to ATO-induced apoptosis via an oxidative-nitrosative mechanism. Intracellular glutathione depletion and lipid peroxidation have been identified as the early cascade of events preceding apoptosis via cytochrome c release and the severe drop of mitochondrial membrane potential (MMP). Conversely, Hep-J5 cells, with normoxic coexpression of HIF-1α and GRP78, were resistant to ATO-induced apoptosis. GRP78-silenced Hep-J5 cells remained resistant to ATO treatment. In contrast, ATO resistance in Hep-J5 cells was overcome by the addition of YC-1, a HIF-1α inhibitor.ConclusionsHIF-1α was identified as the major positive modifier for ATO resistance acquisition in HCC, and it represents a prime molecular target for overcoming ATO resistance.

Effectiveness of esophagectomy in patients with thoracic esophageal squamous cell carcinoma receiving definitive radiotherapy or concurrent chemoradiotherapy through intensity-modulated radiation therapy techniques.

Few large, prospective, randomized studies have investigated the effectiveness of esophagectomy in patients with thoracic esophageal squamous cell carcinoma (TESCC) who receive definitive radiotherapy (RT) or concurrent chemoradiotherapy (CCRT) through modern, intensity modulated‐RT (IMRT) techniques. The therapeutic effects of esophagectomy in patients with TESCC were evaluated using modern clinical staging and RT techniques and suitable RT doses.