• Asian Pacific Journal of Cancer Prevention
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• v.15 no.23
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• pp.10413-10420
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• 2015

Side effects are an unavoidable consequence of chemotherapy drugs, during which liver injury often takes place. The current study was designed to investigate the protective effect of Astragalus polysaccharides (APS) against the hepatotoxicity induced by frequently-used chemical therapy agents, cyclophosphamide (CTX), docetaxel (DTX) and epirubicin (EPI)) in mice. Mice were divided into five groups, controls, low or high dose groups ($DTX_L$, $CTX_L$, $EPI_L$ or $DTX_H$, $CTX_H$, $EPI_H$), and low or high dose chemotherapeutics+APS groups ($DTX_L$+APS, $CTX_L$+APS, $EPI_L$+APS or $DTX_H$+APS, $CTX_H$+APS, $EPI_H$+APS). Controls were treated with equivalent normal saline for 28 days every other day; low or high dose group were intraperitoneal (i.p) injected with low or high doses of CTX, DTX and EPI for 28 days every other day; low or high dose chemotherapeutics+APS group were separately intraperitoneal (i.p) injected with chemotherapeutics for 28 days every other day and i.p with APS (100 mg/kg) for 7 days continually from the 22th to the 28th days. The body weight, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), histopathological features, and ultrastructure morphological change of liver tissues, protein expression level of caspase-3 were estimated at different time points. With high dose treatment of DTX, CTX and EPI, weight gain was inhibited and serum levels of ALT and AST were significantly increased. Sections of liver tissue showed massive hepatotoxicity in $CTX_H$ group compared to the control group, including hepatic lobule disorder, granular and vacuolar degeneration and necrosis in hepatic cells. These changes were confirmed at ultrastructural level, including obvious pyknosis, heterochromatin aggregation, nuclear membrane resolution, and chondrosome crystal decrease. Western blotting revealed that the protein levels of caspase-3 increased in $CTX_H$ group. The low dose groups exhibited trivial hepatotoxicity. More interestingly, after 100 mg/kg APS, liver injury was redecued not only regarding serum transaminase activities (low or high dose chemotherapeutics+APS group), but also from pathological and ultrastructural changes and the protein levels of caspase-3 ($CTX_H$+APS group). In conclusion, DTX, CTX and EPI induce liver damage in a dose dependent manner, whereas APS exerted protective effects.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.16
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• pp.6829-6835
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• 2014

Chemotherapy is the primary therapy for malignant lymphoma (ML). However, the clinical outcome is still far from satisfactory. Consequently, an understanding of the mechanism of modulating cancer cell invasion, migration and metastasis is important for the development of more effective chemotherapeutic agents. FNC, 2'-deoxy-2'-${\beta}$-fluoro-4'-azidocytidine, a novel cytidine analogue, has demonstrated significantly inhibitory effects on proliferation of several non-Hodgkin lymphoma (NHL) cell lines. A previous study indicated that FNC effectively inhibited the growth of Raji and JeKo-1 cells in dose-time dependent effects with $IC_{50}$ values of $0.2{\mu}M$ and $0.097{\mu}M$, respectively. This study was focused on investigating the anti-invasive properties of FNC on NHL cells and its potential mechanisms of action. Cell adhesion and transwell chamber assays were utilized to investigate the anti-invasive effects of FNC on Raji and JeKo-1 cells. Real-time PCR and Western blotting were employed to qualify the expression of ${\beta}$-catenin, the glycogen synthase kinase-3 beta (GSK-$3{\beta}$), E-cadherin vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). The results revealed that FNC remarkably inhibited the adhesion, migration and invasion of two human aggressive non-Hodgkin lymphoma cell lines in a dose dependent manner. Furthermore, ${\beta}$-catenin, MMP-2, MMP-9, VEGF mRNA and protein levels were decreased after FNC treatment, while GSK-$3{\beta}$ and E-cadherin increased. Our studies thus provide evidence and a rationale that FNC may offer an effective chemotherapeutic agent by regulating the invasion and metastasis of aggressive non-Hodgkin lymphoma via inhibition of the Wnt/${\beta}$-catenin signaling pathway.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.16
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• pp.6649-6655
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• 2014

Radiation therapy is an important treatment for head and neck squamous cell carcinoma (HNSCC). However, how to promote radiation sensitivity in HNSCC remains a challenge. This study aimed to investigate the radiosensitizing effects of fenofibrate on HNSCC and explore the underlying mechanisms. HNSCC cell lines CNE-2 and KB were subjected to ionizing radiation (IR), in the presence or absence of fenofibrate treatment. Cell growth and survival, apoptosis and cell cycle were evaluated. In addition, CNE-2 cells were xenografted into nude mice and subjected to IR and/or fenofibrate treatment. The expression of cyclinB and CDK1 was detected by Western blotting. Our results showed that fenofibrate efficiently radiosensitized HNSCC cells and xenografts in mice, and induced apoptosis and G2/M arrest via reducing the activity of the CDK1/cyclinB1 kinase complex. These data suggest that fenofibrate could be a promising radiosensitizer for HNSCC radiotherapy.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.16
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• pp.6737-6743
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• 2014

It is reported that sinomenine (SIN) and 5-fluorouracil (5-FU) both are effective for colon cancer, but their cooperative suppressive effects and toxicity remain to be clarified in detail. This study aimed to determine suppressive effects and toxicity of sinomenine (SIN) plus 5-fluorouracil (5-FU) on LoVo colon carcinoma cells in vitro and in vivo. CCK-8, Hoechst 33258 staining and an annexin V-FITC/PI apoptosis kit were used to detect suppressive effects. Western blotting was applied to investigate the essential mechanism underlying SIN and 5-FU-induced apoptosis. SIN or 5-FU or both were injected into nude mice, and then suppressive effects and side effects were observed. SIN plus 5-FU apparently inhibited the proliferation of LoVo cells and induced apoptosis. Moreover the united effects were stronger than individually (p<0.05). The results of annexin V-FITC/PI staining and Hoechst 33258 staining showed that the percentage of apoptotic cells induced by SIN and 5-FU combined or alone was significantly higher than the control group (p<0.05). Expression of Bax and Bcl-2 was up-regulated and down-regulated respectively. SIN or 5-FU significantly inhibited effects on the volume of tumour xenografts and their combined suppressive effects were stronger (p<0.05). No obvious side effects were observed. It was apparent that the united effects of SIN and 5-FU on the growth of colorectal carcinoma LoVo cells in vitro and in vivo were superior to those using them individually, and it did not markedly increase the side effects of chemotherapy.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.6
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• pp.2635-2640
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• 2014

Sera of cancer patients may contain antibodies that react with a unique group of autologous cellular antigens called tumor-associated antigens (TAAs). The present study aimed to determine whether a mini-array of multiple TAAs would enhance antibody detection and be a useful approach in esophageal cancer detection and diagnosis. Our mini-array of multiple TAAs consisted of eleven antigens, p53, pl6, Impl, CyclinB1, C-myc, RalA, p62, Survivin, Koc, CyclinD1 and CyclinE full-length recombinant proteins. Enzyme-linked immunosorbent assays (ELISA) were used to detect autoantibodies against eleven selected TAAs in 174 sera from patients with esophageal cancer, as well as 242 sera from normal individuals. In addition, positive results of ELISA were confirmed by Western blotting. In a parallel screening trial, with the successive addition of antigen to a final total of eleven TAAs, there was a stepwise increase in positive antibody reactions. The eleven TAAs were the best parallel combination, and the sensitivity and specificity in diagnosing esophageal cancer was 75.3% and 81.0%, respectively. The positive and negative predictive values were 74.0% and 82.0%, respectively, indicating that the parallel assay of eleven TAAs raised the diagnostic precision significantly. In addition, the levels of antibodies to seven antigens, comprising p53, Impl, C-myc, RalA, p62, Survivin, and CyclinD1, were significantly different in various stages of esophageal cancer, which showed that autoantibodies may be involved in the pathogenesis and progression of esophageal cancer. All in all, this study further supports our previous hypothesis that a combination of antibodies might acquire higher sensitivity for the diagnosis of certain types of cancer. A customized mini-array of multiple carefully-selected TAAs is able to enhance autoantibody detection in the immunodiagnosis of esophageal cancer and autoantibodies to TAAs might be reference indicators of clinical stage.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.3
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• pp.1285-1290
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• 2014

The aim was to determine whether ultrasound targeted microbubble destruction (UTMD) promotes dual targeting of HSP72 and HSC70 for therapy of castration-resistant prostate cancer (CRPC), to improve the specific and efficient delivery of siRNA, to induce tumor cell specific apoptosis, and to find new therapeutic targets specific of CRPC.VCaP cells were transfected with siRNA oligonucleotides. HSP70, HSP90 and cleaved caspase-3 expression were determined by real-time quantitative polymerase chain reaction and Western blotting. Apoptosis and transfection efficiency were assessed by flow cytometry. Cell viability assays were used to evaluate safety. We found HSP72, HSC70 and HSP90 expression to be absent or weak in normal prostate epithelial cells (RWPE-1), but uniformly strong in prostate cancerous cells (VCaP). UTMD combined with dual targeting of HSP72 and HSC70 siRNA improve the efficiency of transfection, cell uptake of siRNA, downregulation of HSP70 and HSP90 expression in VCaP cells at the mRNA and protein level, and induction of extensive tumor-specific apoptosis. Cell counting kit-8 assays showed decreased cellular viability in the HSP72/HSC70-siRNA silenced group. These results suggest that the combination of UTMD with dual targeting HSP70 therapy for PCa may be most efficacious, providng a novel, reliable, non-invasive, safe targeted approach to improve the specific and efficient delivery of siRNA, and achieve maximal effects.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.6
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• pp.2753-2758
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• 2014

Background: GC-binding factor 2 (GCF2) is a transcriptional regulator that represses transcriptional activity of the epidermal growth factor receptor (EGFR) by binding to a specific GC-rich sequence in the EGFR gene promoter. In addition to this function, GCF2 has also been identified as a tumor-associated antigen and regarded as a potentially valuable serum biomarker for early human hepatocellular carcinoma (HCC) diagnosis. GCF2 is high expressed in most HCC tissues and cell lines including HepG2. This study focused on the influence of GCF2 on cell proliferation and apoptosis in HepG2 cells. Materials and Methods: GCF2 expression at both mRNA and protein levels in HepG2 cells was detected with reverse transcription (RT) PCR and Western blotting, respectively. RNA interference (RNAi) technology was used to knock down GCF2 mRNA and protein expression. Afterwards, cell viability was analyzed with a Cell Counting Kit-8 (CCK-8), and cell apoptosis and caspase 3 activity by flow cytometry and with a Caspase 3 Activity Kit, respectively. Results: Specific down-regulation of GCF2 expression caused cell growth inhibition, and increased apoptosis and caspase 3 activity in HepG2 cells. Conclusions: These primary results suggest that GCF2 may influence cell proliferation and apoptosis in HepG2 cells, and also provides a molecular basis for further investigation into the possible mechanism at proliferation and apoptosis in HCC.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.12
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• pp.5001-5007
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• 2014

The acetyltransferase inhibitor garcinol, a polyisoprenylated benzophenone, is extracted from the rind of the fruit of Garcinia indica, a plant found extensively in tropical regions. Anti-cancer activity has been suggested but there is no report on its action via inhibiting acetylation against cell proliferation, cell cycle progression, and apoptosis-inhibtion induced by estradiol ($E_2$) in human breast cancer MCF-7 cells. The main purposes of this study were to investigate the effects of the acetyltransferase inhibitor garcinol on cell proliferation, cell cycle progression and apoptosis inhibition in human breast cancer MCF-7 cells treated with estrogen, and to explore the significance of changes in acetylation levels in this process. We used a variety of techniques such as CCK-8 analysis of cell proliferation, FCM analysis of cell cycling and apoptosis, immunofluorescence analysis of NF-${\kappa}B$/p65 localization, and RT-PCR and Western blotting analysis of ac-H3, ac-H4, ac-p65, cyclin D1, Bcl-2 and Bcl-xl. We found that on treatment with garcinol in MCF-7 cells, $E_2$-induced proliferation was inhibited, cell cycle progression was arrested at G0/G1 phase, and the cell apoptosis rate was increased. Expression of ac-H3, ac-H4 and NF-${\kappa}B$/ac-p65 proteins in $E_2$-treated MCF-7 cells was increased, this being inhibited by garcinol but not ac-H4.The nuclear translocation of NF-${\kappa}B$/p65 in $E_2$-treated MCF-7 cells was also inhibited, along with cyclin D1, Bcl-2 and Bcl-xl in mRNA and protein expression levels. These results suggest that the effect of $E_2$ on promoting proliferation and inhibiting apoptosis is linked to hyperacetylation levels of histones and nonhistone NF-${\kappa}B$/p65 in MCF-7 cells. The acetyltransferase inhibitor garcinol plays an inhibitive role in MCF-7 cell proliferation promoted by $E_2$. Mechanisms are probably associated with decreasing ac-p65 protein expression level in the NF-${\kappa}B$ pathway, thus down-regulating the expression of cyclin D1, Bcl-2 and Bcl-xl.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.12
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• pp.4951-4956
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• 2014

Purpose: To investigate IQGAP1 and IQGAP2 expression in hepatocellular carcinoma (HCC) and itsassociation with HCC clinicopathological characteristics and survival outcomes. Methods: IQGAP1 and IQGAP2 mRNA and protein were measured in HCC tissues, para-tumor tissues and normal tissues by RT-PCR and Western blotting. We further examined 150 HCC samples with adjacent para-tumor tissues and 11 normal specimens by immunohistochemistry to evaluate the correlation of IQGAP1 and IQGAP2 with clinicopathological features and prognosis. Results: IQGAP1 mRNA and protein were up-regulated while IQGAP2 mRNA and protein were down-regulated in human HCC tissues compared with para-tumor and normal liver tissues (p<0.05). IQGAP1 expression was higher in primary HCC (122/150, 81.3%) than matched adjacent tissues (30/150, 20%, p<0.001), whereas IQGAP2 was lower (31/150, 20.7% as compared to 112/150, 74.7%, P<0.001). Positive IQGAP1 expression correlated with larger tumor size (p=0.002), advanced TNM stage (p=0.002) and tumor differentiation (III and IV, p=0.034). Negative IQGAP2 expression was significantly associated with larger tumor size (p=0.009), multicentric tumor occurrence (p=0.01), advanced TNM stage (0.009) and tumor differentiation (III and IV, p=0.020). Survival analysis revealed that patients with either IQGAP1+ or IQGAP2-tumors had significantly reduced disease-free survival (p<0.001 and 0.006 respectively) and overall survival (p<0.001 for both). Multivariate analysis showed that IQGAP1/2 switch was an independent prognosis factor for disease-free survival (HR=2.824) and overall survival (HR=2.189). Conclusion: Positive IQGAP1 and negative IQGAP2 expression were closely correlated with tumor progression and could be used as adjunctive biomarkers to improve prognostication for HCC patients.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7179-7186
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• 2013

Background and Aims: Scutellaria is one of the most popular traditional Chinese herbal remedies against various human diseases, including cancer. In this study, we examined the active effects of Scutellaria extract and its main flavonoid constituents on the proportion of side population cells within human multiple myeloma cell line RPMI8226 in vitro and explored the potential molecular mechanisms involved. Materials and Methods: The contents of flavonoids in ethanolic extract of Scutellaria baicalensis Georgi were determined using high performance liquid chromatography. The antiproliferative effect of the ethanolic extract on RPMI-8226 was determined by CCK assay. Apoptosis was measured by annexin combining with propidium iodide in a flow cytometer. Cell cycle analysis was performed by propidium iodide staining in combination with flow cytometry analysis. Hoechst 33342 exclusion assay was used for the identification of side population within RPMI8226 cells. The expression of ABCG2 protein was assessed by Western blotting assay. Results: The content of major flavonoids constitutents of Scutellaria extract was baicalin (10.2%), wogonoside (2.50%), baicalein (2.29%), and wogonin (0.99%), respectively. The crude Scutellaria extract did not show significant anti-proliferative effect, apoptosis induction and cell cycle arrest in RPMI-8226 within the concentrations of $1-75{\mu}g/mL$. However, the ethanolic extract, baicalein, wogonin and baicalin reduced the side population cells in RPMI-8226, and data showed that baicalein and wogonin had stronger inhibitory effects. Correspondingly, they also exhibited significant effects on decreasing the expression level of ABCG2 protein in RPMI-8226 in vitro. Conclusions: Our results for the first time demonstrated a novel mechanism of action for Scutellaria extract and its main active flavonoids, namely targeting SP cells by modulating the expression of ABCG2 protein. This study provides an insight for new therapeutic strategies targeting cancer stem cells of multiple myeloma.