Comparison of Quality of Life Between Breast Cancer Patients Treated With and Without Adjunctive Traditional Chinese Medicine in Taiwan.

In Taiwan, breast cancer has the highest incidence among all cancers. Although adjunctive traditional Chinese medicine treatment (TCM) have been used to ameliorate the side effects or discomfort caused by cancer treatments, no study has focused on the assessment of the quality of life of patients undergoing adjunctive TCM treatments. This study compared the quality of life between breast cancer patients treated with and without adjunctive TCM. Questionnaires were collected from 7 hospitals with a Chinese medicine clinic in 2018 to 2019. Breast cancer patients who had cancer stages I, II, or III and also underwent resection surgery were included in the study. They were divided into 2 groups: patients receiving cancer treatments with adjunctive traditional Chinese medicine (TCM group) and those receiving cancer treatments without adjunctive traditional Chinese medicine (non-TCM group). A 1:1 matching was used to obtain the study participants. The EQ-5D questionnaire was used to assess the quality of life. Statistical analysis was performed using the t-test and ANOVA to compare the differences between variables. The conditional multiple regression model was applied to explore the factors associated with quality of life in breast cancer patients. A total of 543 participants were surveyed, and 450 participants were included in the study. The EQ-5D score of the TCM group (81.60 ± 11.67) was significantly higher than that of the non-TCM group (78.80 ± 13.10; P < .05). The results of a conditional multiple regression model showed that the TCM group had a higher (3.45 points) quality of life than non-TCM group (P = .002) after adjusting for other related factors. After stratifying by cancer stage, patients with cancer stages II and III scored 5.58 and 4.35 points higher in the TCM group than did those in the non-TCM group (P < .05). Breast cancer patients undergoing cancer treatment with adjunctive traditional Chinese medicine have a higher quality of life than those treated without adjunctive traditional Chinese medicine.

Cetyltrimethylammonium Bromide Disrupts Mesenchymal Characteristics of Human Tongue Squamous Cell Carcinoma SCC4 Cells Through Modulating Canonical TGF-ß/Smad/miR-181b/TIMP3 Signaling Pathway.

BACKGROUND/AIM: This study investigated the anti-metastatic effects of cetyltrimethylammonium bromide (CTAB) on tongue squamous cell carcinoma (TSCC) SCC4 cells. MATERIALS AND METHODS: Cell morphology, viability, cell cycle distribution, adhesion, migration, invasion and the expression levels of associated proteins were examined using microscopy, WST-1, wound-healing, Boyden chamber assays, and western blotting, respectively. RESULTS: CTAB significantly affected SCC4 cell morphology from spindle-shaped to cobblestone-shaped and resulted in loss of adherence. CTAB significantly inhibited cell adhesion, migration, and invasion of SCC4 cells, independent of cell viability. CTAB reduced expression of matrix metalloproteinases (MMPs) such as MMP3, MMP7, and MMP14 in a concentration-dependent manner, while it increased expression of tissue inhibitors of metalloproteinase 3 (TIMP3). In addition, CTAB reduced the phosphorylation of mothers against decapentaplegic homolog 2/3 (Smad2/3) proteins, which mediated CTAB-inhibited migration and invasion in SCC4 cells. These effects were reversed by TGF-ß1. CONCLUSION: CTAB attenuates the mesenchymal characteristics through upregulation of TIMP3 by inhibiting the canonical TGF-ß/Smad/miR-181b/TIMP3 signaling involved in extracellular matrix remodeling in SCC4 cells and might be a promising anti-metastatic therapeutic agent for TSCC.

RET Regulates Human Medullary Thyroid Cancer Cell Proliferation through CDK5 and STAT3 Activation.

Medullary thyroid cancer (MTC) is a neuroendocrine tumor that arises from the parafollicular C-cells, which produces the hormone calcitonin. RET is a transmembrane receptor protein-tyrosine kinase, which is highly expressed in MTC. Our previous studies reported that cyclin-dependent kinase 5 (CDK5) plays a crucial role in cancer progression, including MTC. However, the role of CDK5 in GDNF-induced RET signaling in medullary thyroid cancer proliferation remains unknown. Here, we investigated RET activation and its biochemically interaction with CDK5 in GDNF-induced medullary thyroid cancer proliferation. Our results demonstrated that GDNF stimulated RET phosphorylation and thus subsequently resulted in CDK5 activation by its phosphorylation. Activated CDK5 further caused STAT3 activation by its specific phosphorylation at Ser727. Moreover, we also found that GDNF treatment enhanced ERK1/2 and EGR1 activity, which is involved in p35 activation. Interestingly, we identified for the first time that CDK5 physically interacted with RET protein in MTC. Overall, our results provide a new mechanism for medullary thyroid cancer cell proliferation, suggesting that targeting CDK5 may be a promising therapeutic candidate for human medullary thyroid cancer in the near future.

Cetyltrimethylammonium Bromide Disrupts the Mesenchymal Characteristics of HA22T/VGH Cells Via Inactivation of c-Met/PI3K/Akt/mTOR Pathway.

BACKGROUND/AIM: Hepatocellular carcinoma (HCC) arises from hepatocytes, and is the most frequently occurring malignancy of primary liver cancer. In this study, we investigated the anti-metastatic effects of the quaternary ammonium compound, cetyltrimethylammonium bromide (CTAB), on HA22T/VGH HCC cells. MATERIALS AND METHODS: According to our preliminary data, the effect of CTAB on cell cycle distribution, migration, invasion and the associated protein levels was examined using flow cytometry, wound-healing migration, Matrigel transwell invasion assay and western blotting under sub-lethal concentrations. RESULTS: CTAB treatment of HA22T/VGH cells casued dose-dependent mesenchymal-epithelial transition (MET)-like changes and impaired migration and invasion capabilities. In addition, CTAB reduced the levels of metastasis-related proteins including c-Met, phosphoinositide 3-kinase (PI3K), Akt, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), Twist, N-cadherin, and Vimentin. Moreover, pretreatment with hepatocyte growth factor (HGF) rescued CTAB-mediated effects. CONCLUSION: CTAB exhibited potent anti-EMT and anti-metastatic activities through the inhibition of migration and invasion of HA22T/VGH cells. CTAB interrupted the mesenchymal characteristics of HA22T/VGH cells, which were significantly alleviated by HGF in a dose-dependent manner. CTAB has the potential to evolve as a therapeutic agent for HCC.

Future Aspects of CDK5 in Prostate Cancer: From Pathogenesis to Therapeutic Implications.

Cyclin-dependent kinase 5 (CDK5) is a unique member of the cyclin-dependent kinase family. CDK5 is activated by binding with its regulatory proteins, mainly p35, and its activation is essential in the development of the central nervous system (CNS) and neurodegeneration. Recently, it has been reported that CDK5 plays important roles in regulating various biological and pathological processes, including cancer progression. Concerning prostate cancer, the androgen receptor (AR) is majorly involved in tumorigenesis, while CDK5 can phosphorylate AR and promotes the proliferation of prostate cancer cells. Clinical evidence has also shown that the level of CDK5 is associated with the progression of prostate cancer. Interestingly, inhibition of CDK5 prevents prostate cancer cell growth, while drug-triggered CDK5 hyperactivation leads to apoptosis. The blocking of CDK5 activity by its small interfering RNAs (siRNA) or Roscovitine, a pan-CDK inhibitor, reduces the cellular AR protein level and triggers the death of prostate cancer cells. Thus, CDK5 plays a crucial role in the growth of prostate cancer cells, and AR regulation is one of the important pathways. In this review paper, we summarize the significant studies on CDK5-mediated regulation of prostate cancer cells. We propose that the CDK5-p35 complex might be an outstanding candidate as a diagnostic marker and potential target for prostate cancer treatment in the near future.

Myeloid Zinc Finger 1 (MZF1) Maintains the Mesenchymal Phenotype by Down-regulating IGF1R/p38 MAPK/ERα Signaling Pathway in High-level MZF1-expressing TNBC cells.

BACKGROUND/AIM: Signaling regulation of myeloid zinc finger 1 (MZF1) has been implicated in the progression of many human malignancies; however, the mechanistic action of MZF1 in triple-negative breast cancer (TNBC) progression remains elusive. In this study, the aim was to investigate the molecular mechanisms of MZF1 and its functional role in TNBC cellular migration and invasion. MATERIALS AND METHODS: Hs578T and MDA-MB-231 cells were transfected to stably express the acidic domain of MZF1 (MZF160-72), or were transfected with MZF1-specific or ELK1-specific short hairpin RNA (shRNA). Changes in cell morphology and distributions of cellular proteins were observed and subsequently migration and invasion were measured by wound healing and transwell assays. Expression levels of epithelial-mesenchymal transition (EMT)-related genes were carried out using immunoblotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays. Data of transcriptional regulation were obtained from promoter-luciferase reporter and chromatin immunoprecipitation (ChIP) assays. RESULTS: Herein, we found that MZF1 in high-level MZF1-expressing TNBC cells is associated with cell migration, invasion, and mesenchymal phenotype. MZF1 interacted with the promoter region of insulin-like growth factor 1 receptor (IGF1R) to drive invasion and metastasis of high-level MZF1-expressing TNBC cells. Exogenous expression of the acidic domain of MZF1 repressed the binding of endogenous MZF1 to IGF1R promoter via blocking the interaction with ETS-like gene 1 (ELK1). This blockage not only caused MZF1 protein degradation, but also restrained ELK1 nuclear localization in high-level MZF1-expressing TNBC cells. MZF1, but not ELK1, was necessary for the retention of mesenchymal phenotype by repressing IGF1R promoter activity in TNBC cells expressing high levels of MZF1. Activation of the IGF1R-driven p38MAPK-ERα-slug-E-cadherin signaling axis mediated the conversion of mesenchymal cell to epithelial phenotype, caused by MZF1 destabilization. These results suggest that MZF1 is an oncogenic inducer. CONCLUSION: Blocking of the MZF1/ELK1 interaction to reduce MZF1 protein stability by saturating the endogenous MZF1/ELK1 binding domains might be a promising therapeutic strategy for the treatment of high-level MZF1-expressing TNBC.

Arecoline Promotes Migration of A549 Lung Cancer Cells through Activating the EGFR/Src/FAK Pathway.

Arecoline is the primary alkaloid in betel nuts, which are known as a risk factor for oral submucosal fibrosis and oral cancer. Lung cancer is a severe type of carcinoma with high cell motility that is difficult to treat. However, the detailed mechanisms of the correlation between Arecoline and lung cancer are not fully understood. Here, we investigated the effect of Arecoline on migration in lung cancer cell lines and its potential mechanism through the muscarinic acetylcholine receptor 3 (mAChR3)-triggered EGFR/Src/FAK pathway. Our results indicate that different concentrations of Arecoline treatment (10 µM, 20 µM, and 40 µM) significantly increased the cell migration ability in A549 and CL1-0 cells and promoted the formation of the filamentous actin (F-actin) cytoskeleton, which is a crucial element for cell migration. However, migration of H460, CL1-5, and H520 cell lines, which have a higher migration ability, was not affected by Arecoline treatment. The EGFR/c-Src/Fak pathway, which is responsible for cell migration, was activated by Arecoline treatment, and a decreased expression level of E-cadherin, which is an epithelial marker, was observed in Arecoline-treated cell lines. Blockade of the EGFR/c-Src/Fak pathway with the inhibitors of EGFR (Gefitinib) or c-Src (Dasatinib) significantly prevented Arecoline-promoted migration in A549 cells. Gefitinib or Dasatinib treatment significantly disrupted the Arecoline-induced localization of phospho-Y576-Fak during focal adhesion in A549 cells. Interestingly, Arecoline-promoted migration in A549 cells was blocked by a specific mAChR3 inhibitor (4-DAMP) or a neutralizing antibody of matrix metalloproteinase (MMP7 or Matrilysin). Taken together, our findings suggest that mAChR3 might play an essential role in Arecoline-promoted EGFR/c-Src/Fak activation and migration in an A549 lung cancer cell line.

Isochlorogenic Acid C Reverses Epithelial-Mesenchymal Transition via Down-regulation of EGFR Pathway in MDA-MB-231 cells.

BACKGROUND/AIM: Epidermal growth factor receptor (EGFR) has been suggested to play an important role in survival, proliferation, migration, differentiation, and tumorigenesis of many cell types. Breast cancer patients with high EGFR expression have a poor prognosis. In this study, we investigated the molecular mechanism of the inhibitory effect of isochlorogenic acid c (ICAC) extracted from Lonicera japonica on elevated EGFR levels of the triple-negative breast cancer (TNBC) cell line, MDA-MB-231. MATERIALS AND METHODS: The cell viability and cell-cycle analysis were evaluated using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay and flow cytometry, respectively. The migration ability and invasiveness of ICAC-treated MDA-MB-231 were examined by migration and Matrigel invasion assay. The epithelial-mesenchymal-transition (EMT)-related protein expression was examined by western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: ICAC led to significant morphological changes and suppressed migration and invasion capacities of highly metastatic MDA-MB-231 cells. Western blot analysis for EGFR/EMT-associated proteins suggested that ICAC attenuated the mesenchymal traits as observed by up-regulation of epithelial markers and down-regulation of mesenchymal markers as well as decreased activities of matrix metalloproteinase-9 (MMP-9). CONCLUSION: These results suggested that the inhibitory effects of ICAC against EGFR-induced EMT and MDA-MB-231 cell invasion were dependent on the EGFR/ phospholipase Cγ (PLCγ)/extracellular regulated protein kinase ½ (ERK½)/slug signaling pathway. Therefore, the obtained results could provide us clues for the next therapeutic strategy in the treatment of TNBC.

Cdk5 Directly Targets Nuclear p21CIP1 and Promotes Cancer Cell Growth.

The significance of Cdk5 in cell-cycle control and cancer biology has gained increased attention. Here we report the inverse correlation between the protein levels of Cdk5 and p21CIP1 from cell-based and clinical analysis. Mechanistically, we identify that Cdk5 overexpression triggers the proteasome-dependent degradation of p21CIP1 through a S130 phosphorylation in a Cdk2-independent manner. Besides, the evidence from cell-based and clinical analysis shows that Cdk5 primarily regulates nuclear p21CIP1 protein degradation. S130A-p21CIP1 mutant enables to block either its protein degradation or the increase of cancer cell growth caused by Cdk5. Notably, Cdk5-triggered p21CIP1 targeting primarily appears in S-phase, while Cdk5 overexpression increases the activation of Cdk2 and its interaction with DNA polymerase δ. The in vivo results show that Cdk2 might play an important role in the downstream signaling to Cdk5. In summary, these findings suggest that Cdk5 in a high expression status promotes cancer growth by directly and rapidly releasing p21CIP1-dependent cell-cycle inhibition and subsequent Cdk2 activation, which illustrates an oncogenic role of Cdk5 potentially applied for future diagnosis and therapy. Cancer Res; 76(23); 6888-900. ©2016 AACR.

MZF-1/Elk-1 interaction domain as therapeutic target for protein kinase Cα-based triple-negative breast cancer cells.

Recent reports demonstrate that the expression of protein kinase C alpha (PKCα) in triple-negative breast cancer (TNBC) correlates with decreased survival outcomes. However, off-target effects of targeting PKCα and limited understanding of the signaling mechanisms upstream of PKCα have hampered previous efforts to manipulate this ubiquitous gene. This study shows that the expression of both myeloid zinc finger 1 (MZF-1) and Ets-like protein-1 (Elk-1) correlates with PKCα expression in TNBC. We found that the acidic domain of MZF-1 and the heparin-binding domain of Elk-1 facilitate the heterodimeric interaction between the two genes before the complex formation binds to the PKCα promoter. Blocking the formation of the heterodimer by transfection of MZF-160-72 or Elk-1145-157 peptide fragments at the MZF-1 / Elk-1 interface decreases DNA-binding activity of the MZF-1 / Elk-1 complex at the PKCα promoter. Subsequently, PKCα expression, migration, tumorigenicity, and the epithelial-mesenchymal transition potential of TNBC cells decrease. These subsequent effects are reversed by transfection with full-length PKCα, confirming that the MZF-1/Elk-1 heterodimer is a mediator of PKCα in TNBC cells. These data suggest that the next therapeutic strategy in treating PKCα-related cancer will be developed from blocking MZF-1/Elk-1 interaction through their binding domain.

Suppression of breast cancer cell growth by Her2-reduced AR serine 81 phosphorylation.

Breast cancer is a hormone-related carcinoma and the most commonly diagnosed malignancy in women. Although Her-2, estrogen receptor (ER), and progesterone receptor (PR) are the major diagnostic markers and therapeutic targets to breast cancer, searching for additional molecular targets remains an important issue and one of the candidates is androgen receptor (AR). AR has been shown expressed in 70% breast cancer patients and connects to low recurrence and high survival rate. Our previous study demonstrates that Ser81 phosphorylation of AR in prostate cancer cells is critical for its protein stability modulated by human epidermal growth factor receptor-2 (Her2). The aim of this study is to investigate the influence of Her2 and AR in proliferation of breast cancer cell line, MDA-MB-453. The data show that AR which was activated by synthetic androgen R1881 suppressed the proliferation of MDA-MB-453 cells. Notably, AR activation decreased the protein levels of cell growth-related proteins, including cyclin A, cyclin B, and early growth response protein 1 (Egr1), while cell-cycle inhibitor protein p27 was increased. Besides, Heregulin (HRG)-induced Her2 activation decreased the AR protein levels and its Ser81 phosphorylation. Her2 small molecular inhibitor, Lapatinib, dose-dependently suppressed cell proliferation while the levels of phospho-Ser81 AR and p27 protein were increased. Phospho-Ser81 AR was also increased after Her2 knockdown. Specifically, the influence of phospho-Ser81 AR by Lapatinib was primarily found in the nucleus of MDA-MD-453 cells, where the cell proliferation might directly be interfered. In conclusion, our findings indicate that Her2 might negatively regulate AR phosphorylation/activation and contribute to regulate the proliferation of MDA-MB 453 cells.

Protein kinase C α is involved in the regulation of AXL receptor tyrosine kinase expression in triple-negative breast cancer cells.

AXL receptor tyrosine kinase is overexpressed in triple-negative breast cancer (TNBC), and has a function in cancer progression and metastases. However, the mechanism underlying AXL gene regulation in TNBC remains unknown. In this study, the involvement of protein kinase C α (PKCα) in the expression of AXL was investigated in human TNBC cells. The microarray data from other studies showed that PKCα is significantly correlated with AXL expression in TNBC cell lines. Tissue array analysis also confirmed their correlation in TNBC. The PKCα inhibitor Go6976 was used to treat MDA­MB­231 and Hs578T TNBC cells, which resulted in decreased expression of AXL and epithelia-mesenchymal transition-related gene vimentin, and decreased cell proliferation. An MZF­1 acidic domain fragment (MZF-1 peptide), which was designed to downregulate PKCα expression, was transfected into the cells and resulted in inhibition of AXL expression. This effect was reversed by co­treatment with the constitutive form of PKCα. Moreover, the downregulation of PKCα was also confirmed by treatment with TAT­fused MZF­1 peptide. Thus, the current study proposes that AXL may be correlated with PKCα­dependent TNBC cells, and could be modulated by MZF­1 peptides.

Anemone altaica Induces Apoptosis in Human Osteosarcoma Cells.

In the past decade, no significant improvement has been made in chemotherapy for osteosarcoma (OS). To develop improved agents against OS, we screened 70 species of medicinal plants and treated two human OS cell lines with different agent concentrations. We then examined cell viability using the MTT assay. Results showed that a candidate plant, particularly the rhizomes of Anemone altaica Fisch. ex C. A. Mey aqueous extract (AAE), suppressed the viability of HOS and U2OS cells in a concentration-dependent manner. Flow cytometry analysis revealed that AAE significantly increased the amount of cell shrinkage (Sub-G1 fragments) in HOS and U2OS cells. Moreover, AAE increased cytosolic cytochrome c and Bax, but decreased Bcl-2. The amount of cleaved caspase-3 and poly-(ADP-ribose) polymerase-1 (PARP-1) were significantly increased. AAE suppressed the growth of HOS and U2OS through the intrinsic apoptotic pathway. Data suggest that AAE is cytotoxic to HOS and U2OS cells and has no significant influence on human osteoblast hFOB cells. The high mRNA levels of apoptosis-related factors (PPP1R15A, SQSTM1, HSPA1B, and DDIT4) and cellular proliferation markers (SKA2 and BUB1B) were significantly altered by the AAE treatment of HOS and U2OS cells. Results show that the anticancer activity of AAE could up-regulate the expression of a cluster of genes, especially those in the apoptosis-related factor family and caspase family. Thus, AAE has great potential as a useful therapeutic drug for human OS.

MZF-1/Elk-1 Complex Binds to Protein Kinase Cα Promoter and Is Involved in Hepatocellular Carcinoma.

In this study, the molecular mechanism of protein kinase C alpha (PKCα) gene regulation in hepatocellular carcinoma (HCC) involving Ets-like protein-1 (Elk-1) and myeloid zinc finger-1 (MZF-1) was investigated. The luciferase reporter assay results revealed that the presence of both MZF-1 and Elk-1 significantly contributed to the upregulation of PKCα gene transcription activity, and the transcriptional activity decreased when the transfection included a DNA-binding-deficient (∆DBD) gene vector of either MZF-1 or Elk-1 DNA-binding deficiency (MZF-1∆DBD or Elk-1∆DBD), thereby indicating that the enhanced expression of PKCα was caused by the binding of MZF-1 and/or Elk-1 with the PKCα promoter. We investigated MZF-1 and Elk-1 to determine whether they bind to each other. The results of immunoprecipitation (IP), Co-IP, chromatin IP (ChIP), and Re-ChIP analyses indicated that Elk-1 can directly bind to the N-terminal region of MZF-1 and MZF-1 can directly bind to the C-terminal region of Elk-1 to form a complex before attaching to the PKCα promoter. Furthermore, when MZF-1∆DBD or Elk-1∆DBD was added to the cells, PKCα expression decreased, and cell proliferation, migration, invasion, and tumorigenicity also decreased. These findings suggest that PKCα expression in HCC could be stimulated by the formation of MZF-1/Elk-1 complex, which directly binds to the PKCα promoter.

Antitumor activity of acriflavine in lung adenocarcinoma cell line A549.

UNLABELLED: Aim/Materials and Methods: In order to develop better drugs against non-small cell lung cancer (NSCLC), we screened a variety of compounds and treated the human lung adenocarcinoma cell line A549 with different drug concentrations. We then examined the cell viability using the MTT assay. RESULTS: Data show that a new candidate drug, acriflavine (ACF), suppresses the viability of A549 cells in a concentration- and time-dependent manner. Flow cytometry analysis revealed that ACF significantly caused cell growth arrest in the G2/M phase on A549 cells. Moreover, ACF decreased Bcl-2 expression and increased Bax expression. The content of cleaved poly(ADP-ribose)polymerase-1 (PARP-1) and caspase-3 are significantly increased. These findings suggest that ACF is cytotoxic against A549 cells and suppresses A549 cells growth through the caspase-3 activation pathway. In the in vivo test, nude mice bearing A549 cells xenografts by intravenous injection were randomly assigned into two groups: control and experimental group. Treatment was initiated 10 days after implantation and intraperitoneal injection of 0.9% normal saline or 2 mg/kg of ACF was continued daily for five weeks. ACF treatment significantly decreased tumor size and tumor spots on lung surface of tumor-bearing mice. CONCLUSION: ACF can inhibit cell growth in A549 cells. Our results may assist on the delineation of the mechanism(s) leading to NSCLC cell growth inhibition and provide a new antitumor strategy against NSCLC.

Antitumor activity of acriflavine in human hepatocellular carcinoma cells.

Patients suffering from advanced hepatocellular carcinoma can generally be treated only by targeted therapy to achieve a survival rate that lasts a few months more than that achieved with conventional therapy. To develop better drugs against hepatocellular carcinoma, we screened a variety of compounds and treated four human hepatocellular carcinoma (HCC) cell lines with different drug concentrations. We then examined cell viability using the MTT assay. Results show that a new candidate drug, acriflavine (ACF), suppresses the viability of HCC cell lines in a dose-dependent manner. Flow cytometry analysis reveals that ACF significantly induces the accumulation of a Sub-G1 population of Mahlavu cells. Moreover, ACF decreases Bcl-2 expression and caspase-3 activation. The content of cleaved poly-(ADP-ribose)polymerase-1 (PARP-1) is significantly increased. These findings suggest that ACF suppresses HCC cell growth through the caspase-3 activation pathway. Compared to clinically-approved drugs, the IC50 of ACF (1 µM) is nearly ten-fold lower than that of sorafenib (13 µM). In the in vivo test, nude mice received Mahlavu cell xenografts subcutaneously and were randomly assigned into two groups: control and experimental groups. Treatment was initiated 3 days after implantation and intraperitoneal injection of 0.9 % normal saline or 2 mg/Kg of ACF was continued daily for five weeks. Tumors were palpable in vehicle-treated mice by day 3 and grew to approximately 2000 mm3 by the end of the experiment, whereas mice treated with ACF experience tumor growth to approximately 500 mm3. We, thus, suggest that ACF can inhibit cell growth in HCC cells. Our results may assist the delineation of the mechanism(s) leading to HCC cell growth inhibition and provide a new target therapy capable to prolong the survival rate of patients in advanced stage.

Emodin and Aloe-Emodin Suppress Breast Cancer Cell Proliferation through ER α Inhibition.

The anthraquinones emodin and aloe-emodin are abundant in rhubarb. Several lines of evidence indicate that emodin and aloe-emodin have estrogenic activity as phytoestrogens. However, their effects on estrogen receptor α (ER α ) activation and breast cancer cell growth remain controversial. The goal of this study is to investigate the effects and molecular mechanisms of emodin and aloe-emodin on breast cancer cell proliferation. Our results indicate that both emodin and aloe-emodin are capable of inhibiting breast cancer cell proliferation by downregulating ER α protein levels, thereby suppressing ER α transcriptional activation. Furthermore, aloe-emodin treatment led to the dissociation of heat shock protein 90 (HSP90) and ER α and increased ER α ubiquitination. Although emodin had similar effects to aloe-emodin, it was not capable of promoting HSP90/ER α dissociation and ER α ubiquitination. Protein fractionation results suggest that aloe-emodin tended to induce cytosolic ER α degradation. Although emodin might induce cytosolic ER α degradation, it primarily affected nuclear ER α distribution similar to the action of estrogen when protein degradation was blocked. In conclusion, our data demonstrate that emodin and aloe-emodin specifically suppress breast cancer cell proliferation by targeting ER α protein stability through distinct mechanisms. These findings suggest a possible application of anthraquinones in preventing or treating breast cancer in the future.

Expression of protein kinase C α and the MZF-1 and Elk-1 transcription factors in human breast cancer cells.

Recently, our research into hepatocellular carcinoma (HCC) has shown that the transcription factors Myeloid Zinc Finger-1 (MZF-1) and Ets-like-protein 1 are related to protein kinase C alpha (PKCα) expression. The purpose of this study was to determine the correlation of the expression of PKCαwith the expressions of Elk-1 and MZF-1 in various differentiated breast cancer cell lines: MDA- MB-231, Hs57BT, SKBR3, MDA-MB-468 and MCF-7. The malignant potential in the five lines of breast cancer cells was examined by using a cell proliferation/migration/invasion assay and the protein and mRNA levels of PKCα, ElK-1 and MZF-1 were examined by Western blot and RT-PCR analysis, re- spectively. The results showed that there were obvious signs of migration and invasion of cells in MDA- MB-231 and Hs57BT cells, little signs of cell migration and invasion in MDA-MB-468 cells, and no sign in SKBR3 and MCF-7 cells. Moreover, the highest expression levels of PKCα, Elk-1 and MZF-1 were also observed in MDA-MB-231 and Hs57BT cells when compared to the other breast cancer cell lines. These findings confirm that elevated expression of PKCαin breast cancer cells may be correlated with the potential of cell migration and invasion, and suggest an association between the expression of PKCα and the expression of the transcription factors Elk-1 and MZF-1.

A reappraisal of three-marker (ER/Vim/CEA), four-marker (ER/Vim/CEA/PR), and five-marker (ER/Vim/CEA/PR/p16INK4a) panels in the diagnostic distinction between primary endocervical and endometrial adenocarcinomas in a tissue microarray study.

BACKGROUND: The choice of appropriate therapeutic plans for primary endocervical adenocarcinomas (ECA) and endometrial adenocarcinomas (EMA) depend on the tumor's site of origin. The purpose of this study was to compare the performances of the commonly used three-marker (ER/Vim/CEA), four-marker (ER/Vim/CEA/PR) and five-marker (ER/Vim/CEA/PR/p16INK4a) panels in distinguishing between primary ECA and EMA. METHODS: A tissue microarray was constructed using paraffin-embedded, formalin-fixed tissues from 35 hysterectomy specimens, including 14 ECA and 21 EMA. Utilizing the avidin-biotin (ABC) technique, tissue array sections were immunostained with five commercially available antibodies (ER, Vim, CEA, PR and p16INK4a) to evaluate the performances of their respective three-, four- and five-marker panels in distinguishing between primary ECA and EMA. RESULTS: ER, PR and Vim were more likely to be expressed in EMA, while CEA and p16INK4a were frequently expressed in ECA. The three-marker (ER/Vim/CEA) panel exhibits the most favorable performance in the distinction between these two gynecologic malignancies (ECA vs. EMA). CONCLUSION: According to our data, when histomorphological and clinical doubt exists as to the primary site of origin, we recommend that the conventional three-marker (ER/Vim/CEA) panel is sufficient, appropriate and useful in distinguishing between primary ECA and EMA, instead of the four-marker (ER/Vim/CEA/PR) and five-marker (ER/Vim/CEA/PR/p16INK4a) panels. Ancillary PR and p16INK4a add no supplementary value to the performance of the conventional three-marker (ER/Vim/CEA) panel.