In-vivo transfection of pcDNA3.1-IGFBP7 inhibits melanoma growth in mice through apoptosis induction and VEGF downexpression

Background

Genome-wide RNA interference screening study revealed that loss of expression of insulin-like growth factor binding protein 7 (IGFBP7) is a critical step in development of a malignant melanoma (MM), and this secreted protein plays a central role in apoptosis of MM. In this study we constructed pcDNA3.1-IGFBP7 to obtain high expression of IGBPF7 and to inhibit the growth of MM in C57BL/6J mice.

Methods

pcDNA3.1-IGFBP7 was transfected into B16-F10 cell, the expression of IGFBP7 was detected by RT-PCR and western blot. The proliferations and apoptosis rates of transfected and control cells were measured by CCK8 and FCM, respectively. The tumorigenicity and tumor growth in both pcDNA3.1-IGFBP7 group and control groups were studied in C57BL/6J mice model. IGFBP7, caspase-3, and VEGF expressions in tumor tissue were measured by immunohistochemistry. Apoptosis of tumors were detected by TUNEL.

Results

We demonstrated this plasmid inhibited proliferation of B16-F10 melanoma cells efficiently in vivo, exploiting the high expression of IGFBP7. More importantly, in-vivo transfection of pcDNA3.1-IGFBP7 inhibited MM growth in C57BL/6J mice. The inhibition of MM growth was proved owing to apoptosis and reduced expression of VEGF induced by pcDNA3.1-IGFBP7.

Conclusions

These results suggest a potential new clinical strategy for MM gene treatment.     

Fractionated irradiation induced radio-resistant esophageal cancer EC109 cells seem to be more sensitive to chemotherapeutic drugs

Background

Chemo-radiotherapy, a combination of chemotherapy and radiotherapy, is the most frequent treatment for patients with esophageal cancer. In the process of radiotherapy, the radiosensitive cancer will become a radio-resistant one.

Methods

In order to detect the chemotherapeutic drug sensitivity in radio-resistant cancer cells and improve the therapy efficiency, we firstly established a radio-resistant esophageal cancer cell model (referred to as EC109/R) from the human esophageal squamous cell carcinoma cell line EC109 through fractionated irradiation using X-rays. The radio-sensitivity of EC109/R cells was measured by clonogenic assay. To detect the drug sensitivity for EC109/R compared to its parent cells, we employed MTT method to screen the effectiveness of five different drugs commonly used in clinical therapy. The ratio of apoptosis was examined by flow cytometry.

Results

EC109/R cells were more sensitive to 5-fluorouracil, doxorubicin, paclitaxel and etoposide, but tolerant to cisplatin compared to its original cells.

Conclusion

Our study implies that fractionated irradiation induced radio-resistant esophageal cancer cell is more sensitive to certain kind of chemotherapeutic drugs. It provides evidence for choosing the sequence of radiotherapy and chemotherapy in esophageal cancer.     

SULF2 overexpression positively regulates tumorigenicity of human prostate cancer cells

Background

SULF2 is a 6-O-endosulfatase which removes 6-O sulfate residues from N-glucosamine present on heparan sulfate (HS). The sulfation pattern of HS influences signaling events mediated by heparan sulfate proteoglycans (HSPGs) located on cell surface, which are critical for the interactions with growth factors and their receptors. Alterations in SULF2 expression have been identified in the context of several cancer types but its function in cancer is still unclear where the precise molecular mechanism involved has not been fully deciphered. To further investigate SULF2 role in tumorigenesis, we overexpressed such gene in prostate cancer cell lines.

Methods

The normal prostate epithelial cell line RWPE-1 and the prostate cancer cells DU-145, and PC3 were transfected with SULF2-expressing plasmid pcDNA3.1/Myc-His(−)-Hsulf-2. Transfected cells were then submitted to viability, migration and colony formation assays.

Results

Transfection of DU-145 and PC3 prostate cancer cells with SULF2 resulted in increased viability, which did not occur with normal prostate cells. The effect was reverted by the knockdown of SULF2 using specific siRNAs. Furthermore, forced expression of SULF2 augmented cell migration and colony formation in both prostate cell lines. Detailed structural analysis of HS from cells overexpressing SULF2 showed a reduction of the trisulfated disaccharide UA(2S)-GlcNS(6S). There was an increase in epithelial-mesenchymal transition markers and an increase in WNT signaling pathway.

Conclusions

These results indicate that SULF2 have a pro-tumorigenic effect in DU-145 and PC3 cancer cells, suggesting an important role of this enzyme in prostatic cancer metastasis.     

Osthole induces G2/M arrest and apoptosis in lung cancer A549 cells by modulating PI3K/Akt pathway

Background

To explore the effects of Osthole on the proliferation, cell cycle and apoptosis of human lung cancer A549 cells.

Methods

Human lung cancer A549 cells were treated with Osthole at different concentrations. Cell proliferation was measured using the MTT assay. Cell cycle was evaluated using DNA flow cytometry analysis. Induction of apoptosis was determined by flow cytometry and fluorescent microscopy. The expressions of Cyclin B1, p-Cdc2, Bcl-2, Bax, t-Akt and p-Akt were evaluated by Western blotting.

Results

Osthole inhibited the growth of human lung cancer A549 cells by inducing G2/M arrest and apoptosis. Western blotting demonstrated that Osthole down-regulated the expressions of Cyclin B1, p-Cdc2 and Bcl-2 and up-regulated the expressions of Bax in A549 cells. Inhibition of PI3K/Akt signaling pathway was also observed after treating A549 cells with Osthole.

Conclusions

Our findings suggest that Osthole may have a therapeutic application in the treatment of human lung cancer.     

Acquired platinum resistance enhances tumour angiogenesis through angiotensin II type 1 receptor in bladder cancer

Background:

We investigated the changes in reactive oxygen species (ROS) and angiogenesis through angiotensin II (Ang II) type 1 receptor (AT1R) after the development of acquired platinum resistance in bladder cancer.

Methods:

Four invasive human bladder cancer cell lines, T24, 5637, T24PR, and 5637PR, were used in vitro, whereas in vivo, T24 and T24PR cells were used. T24PR and 5637PR cells were newly established at our institution as acquired platinum-resistant sublines by culturing in cisplatin (CDDP)-containing conditioned medium for 6 months.

Results:

Ang II induced significantly higher vascular endothelial growth factor (VEGF) production in T24PR and 5637PR cells than in their corresponding parent cells in vitro, whereas Ang II induced a further increase in VEGF production. These platinum-resistant cells also showed significantly higher AT1R expression than their corresponding parent cells. ROS was also significantly upregulated in T24PR and 5637PR cells, whereas increased AT1R expression was significantly downregulated by scavenging free radicals. We also demonstrated the efficacy of AT1R blockade at suppressing the growth of platinum-resistant xenograft model.

Conclusion:

Our findings indicate a new molecular mechanism for upregulated AT1R signalling through increased ROS when tumours progressed after the CDDP-based regimens, and shed light on the importance of AT1R blockade for platinum-resistant bladder cancers.     

Methylation associated inactivation of RASSF1A and its synergistic effect with activated K-Ras in nasopharyngeal carcinoma

Background

Epigenetic silencing of tumor suppressor genes associated with promoter methylation is considered to be a hallmark of oncogenesis. RASSF1A is a candidate tumor suppressor gene which was found to be inactivated in many human cancers. Although we have had a prelimilary cognition about the function of RASSF1A, the exact mechanisms about how RASSF1A functions in human cancers were largely unknown. Moreover, the effect of mutated K-Ras gene on the function of RASSF1A is lacking. The aim of this study was to investigate the expression profile and methylation status of RASSF1A gene, and to explore its concrete mechanisms as a tumor suppressor gene in Nasopharyngeal Carcinoma.

Methods

We examined the expression profile and methylation status of RASSF1A in two NPC cell lines, 38 primary nasopharyngeal carcinoma and 14 normal nasopharyngeal epithelia using RT-PCR and methylated specific PCR(MSP) respectively. 5-aza-dC was then added to confirm the correlation between hypermethylation status and inactivation of RASSF1A. The NPC cell line CNE-2 was transfected with exogenous pcDNA3.1(+)/RASSF1A plasmid in the presence or absence of mutated K-Ras by liposome-mediated gene transfer method. Flow cytometry was used to examine the effect of RASSF1A on cell cycle modulation and apoptosis. Meanwhile, trypan blue dye exclusion assays was used to detect the effect of RASSF1A transfection alone and the co-transfection of RASSF1A and K-Ras on cell proliferation.

Results

Promoter methylation of RASSF1A could be detected in 71.05% (27/38) of NPC samples, but not in normal nasopharyngeal epithelia. RASSF1A expression in NPC primary tumors was lower than that in normal nasopharyngeal epithelial (p < 0.01). Expression of RASSF1A was down-regulated in two NPC cell lines. Loss of RASSF1A expression was greatly restored by the methyltransferase inhibitor 5-aza-dC in CNE-2. Ectopic expression of RASSF1A in CNE-2 could increase the percentage of G0/G1 phase cells (p < 0.01), inhibit cell proliferation and induce apoptosis (p < 0.001). Moreover, activated K-Ras could enhance the growth inhibition effect induced by RASSF1A in CNE-2 cells (p < 0.01).

Conclusion

Expression of RASSF1A is down-regulated in NPC due to the hypermethylation of promoter. Exogenous expression of RASSF1A is able to induce growth inhibition effect and apoptosis in tumor cell lines, and this effect could be enhanced by activated K-Ras.     

Antisense oligonucleotide targeting Livin induces apoptosis of human bladder cancer cell via a mechanism involving caspase 3

Background and Aim

in recent years, Livin, a new member of IAPs family, is found to be a key molecule in cancers. Researchers consider Livin may become a new target for tumor therapy; however, the role of it in bladder cancer is still unclear. The purpose of this article is to investigate Antisense Oligonucleotide (ASODN) of Livin on treating bladder cancer cell and underlying mechanisms.

Methods

Phosphorathioate modifying was used to synthesize antisense oligonucleotides targeting Livin, followed by transfection into human bladder cancer cell 5637. After transfection, Livin mRNA and protein level, cell proliferation and apoptosis changes, caspase3 level and its effect on human bladder cancer transplantable tumor in nude mice were measured.

Result

results showed Livin ASODN effectively inhibited Livin expression and tumor cell proliferation, and these effects probably through enhanced caspase3 activity and apoptosis of tumor cells. In nude mice transplantable tumor model, Livin expressions were inhibited meanwhile caspase3 expression was increased. Tumor growth slowed down and apoptosis was enhanced.

Conclusion

Our data suggest that Livin plays an important role in inhibiting apoptosis of bladder cancer cells. Livin ASODN may promote cell apoptosis, inhibit bladder cancer growth, and become one of the methods of gene therapy for bladder cancer.     

Reduced association of anti-apoptotic protein Mcl-1 with E3 ligase Mule increases the stability of Mcl-1 in breast cancer cells

Background:

Mechanisms that increase resistance to apoptosis help promote cellular transformation. Cancer cells have deregulated apoptotic pathways, where increased expression and stability of anti-apoptotic proteins Mcl-1 and Bcl-2 increases resistance to apoptosis. Pathways that increase the stability of proteins in cancer cells remain poorly understood.

Methods:

Using human mammary epithelial and established breast cancer cell lines, we assessed the mechanisms that increase the stability of anti-apoptotic proteins in breast cancer cells by caspase assay, western blot, small-inhibitory RNA treatment and immunoprecipitation.

Results:

While breast cancer cells were resistant to de novo inhibition of protein synthesis, a rapid proteosome-mediated degradation of Mcl-1 and Bcl-2 induced apoptosis in mammary epithelial cells. Although Mule, an E3 ligase that targets Mcl-1 for degradation was expressed in mammary epithelial and breast cancer cell lines, rapid increase of polyubiquitinated Mcl-1 and Bcl-2 was detected only in mammary epithelial cells. Only transient formation of the Mule–Mcl-1 complex was detected in breast cancer cells. Downregulation of pERK1/2 in breast cancer cells reduced Mcl-1 levels and increased Mcl-1/Mule complex.

Conclusion:

Our findings suggest that reduced Mule/Mcl-1 complex has a significant role in increasing the stability of Mcl-1 in breast cancer cells and increased resistance to apoptosis.     

Acquisition of epithelial–mesenchymal transition is associated with Skp2 expression in paclitaxel-resistant breast cancer cells

Background:

Breast cancer is the most common female malignant disease, and the second leading cause of cancer-related death in the United States. Acquired resistance to chemotherapeutic drugs is a pivotal reason that leads to worse treatment outcome of breast cancer. Therefore, it is urgent to elucidate the mechanism of drug resistance in breast cancer.

Methods:

To investigate the underlying molecular basis of the acquired resistant cells to paclitaxel in breast cancer, we used multiple methods including real-time RT–PCR, western blotting analysis, migration and invasion assays, wound healing assay, and transfection.

Results:

We found that epithelial–mesenchymal transition (EMT) is involved in paclitaxel-resistant (PR) breast cancer cells. The resistant cells with EMT features exhibit increased migration and invasion activities. Mechanistically, high expression of Skp2 was found to be associated with EMT in PR cells. Notably, depletion of Skp2 in PR cells led to partial reversal of EMT phenotype.

Conclusions:

These findings suggest that Skp2 was critically involved in PR-mediated EMT. Skp2 could be a potential therapeutic target for breast cancer.     

Elevated expression of Dickkopf-1 increases the sensitivity of human glioma cell line SHG44 to BCNU

Background

Studies have shown that Dickkopf-1 (DKK-1) is involved in tumorigenesis. Recently, we found that 9 out of 12 human glioma cell lines had high level of DKK-1 protein while the other 3 had very low or non-detectable level of DKK-1. The aim of this study is to further examine the function of DKK-1 in glioma cells.

Materials and methods

The glioma cell line SHG₄₄ was obtained from a patient with grade II-III astrocytoma. SHG₄₄ cells were transfected with a human DKK-1 gene. Transfection of the empty vector pcDNA3.1 was used as negative control. Sensitivity to BCNU was measured by Annexin-V staining. Expression of bax, bcl-2 and caspase-3 of three groups was determined by immunohistochemistry.

Results

The tranfection was confirmed by PCR, RT-PCR and Western blot. More apoptotic cell death was observed in the DKK-1 transfected cells, comparing to the non-transfected cells, or cells with empty vector. The expression of bax and caspase-3 of the SHG₄₄ -DDK-1 increased, whereas the expression of bcl-2 decreased

Conclusion

Our results indicated that DKK-1 has a pro-apoptotic function of in glioma.     

Effects of ectopic HER-2/neu gene expression on the COX-2/PGE2/P450arom signaling pathway in endometrial carcinoma cells: HER-2/neu gene expression in endometrial carcinoma cells

Objectives

To investigate the role of HER-2/neu-mediated COX-2/P450arom signal in estrogen-dependent endometrial carcinoma.

Methods

The recombinant eukaryotic expression vector, pcDNA3.1-HER-2/neu, was constructed and transfect to Ishikawa endometrial carcinoma cells. The expression of COX-2 and P450arom in transfected cells were detected by real-time PCR and western blotting. The levels of estrogen in cell supernatants were detected by ELISA.

Results

Over-expression of HER-2/neu in transfected cells was confirmed by real-time PCR and western blotting. The levels of autocrine estrogen in transfected cells was significantly increased which combination with the enhancement of COX-2 and P450arom expression in transfected cells.

Conclusion

HER-2/neu induced the improvement of autocrine estrogen in endometrial carcinoma cell through triggering the COX-2/P450arom signal.     

Downregulation of SPARC expression decreases gastric cancer cellular invasion and survival

Background

Secreted protein acidic and rich in cysteine (SPARC) plays a key role in the development of many tissues and organ types. Aberrant SPARC expression was found in a wide variety of human cancers, contributes to tumor development. Because SPARC was found to be overexpressed in human gastric cancer tissue, we therefore to explore the expression of SPARC in gastric cancer lines and the carcinogenic mechanisms.

Methods

SPARC expression was evaluated in a panel of human gastric cancer cell lines. MGC803 and HGC 27 gastric cancer cell lines expressing high level of SPARC were transiently transfected with SPARC-specific small interfering RNAs and subsequently evaluated for effects on invasion and proliferation.

Results

Small interfering RNA-mediated knockdown of SPARC in MGC803 and HGC 27 gastric cancer cells dramatically decreased their invasion. Knockdown of SPARC was also observed to significantly increase the apoptosis of MGC803 and HGC 27 gastric cancer cells compared with control transfected group.

Conclusions

Our data showed that downregulating of SPARC inhibits invasion and growth of human gastric cancer cells. Thus, targeting of SPARC could be an effective therapeutic approach against gastric cancer.     

Mutant p53 mediates survival of breast cancer cells

Background:

p53 is the most commonly mutated tumour-suppressor gene in human cancers. Unlike other tumour-suppressor genes, most p53 cancer mutations are missense mutations within the core domain, leading to the expression of a full-length mutant p53 protein. Accumulating evidence has indicated that p53 cancer mutants not only lose tumour suppression activity but also gain new oncogenic activities to promote tumourigenesis.

Methods:

The endogenous mutant p53 function in human breast cancer cells was studied using RNA interference (RNAi). Gene knockdown was confirmed by quantitative PCR and western blotting. Apoptosis was evaluated by morphological changes of cells, their PARP cleavage and annexin V staining.

Results:

We show that cancer-associated p53 missense mutants are required for the survival of breast cancer cells. Inhibition of endogenous mutant p53 by RNAi led to massive apoptosis in two mutant p53-expressing cell lines, T47D and MDA-MB-468, but not in the wild-type p53-expressing cells, MCF-7 and MCF-10A. Reconstitution of an RNAi-insensitive mutant p53 in MDA-MB-468 cells completely abolished the apoptotic effects after silencing of endogenous mutant p53, suggesting the specific survival effects of mutant p53. The apoptotic effect induced by mutant p53 ablation, however, is independent of p63 or p73 function.

Conclusion:

These findings provide clear evidence of a pro-survival ‘gain-of-function'' property of a subset of p53 cancer mutants in breast cancer cells.     

Silibinin Enhances Ultraviolet B-Induced Apoptosis in MCF-7 Human Breast Cancer Cells

Purpose

Chemotherapies for breast cancer generally have strong cellular cytotoxicity and severe side effects. Thus, significant emphasis has been placed on combinations of naturally occurring chemopreventive agents. Silibinin is a major bioactive flavonolignan extracted from milk thistle with chemopreventive activity in various organs including the skin, prostate, and breast. However, the mechanism underlying the inhibitory action of silibinin in breast cancer has not been completely elucidated. Therefore, we investigated the effect of silibinin in MCF-7 human breast cancer cells and determined whether silibinin enhances ultraviolet (UV) B-induced apoptosis.

Methods

The effects of silibinin on MCF-7 cell viability were determined using the MTT assay. The effect of silibinin on PARP cleavage, as the hallmark of apoptotic cell death, and p53 protein expression in MCF-7 cells was analyzed using Western blot. The effect of silibinin on UVB-induced apoptosis in MCF-7 cells was analyzed by flow cytometry.

Results

A dose- and time-dependent reduction in viability was observed in MCF-7 cells treated with silibinin. Silibinin strongly induced apoptotic cell death in MCF-7 cells, and induction of apoptosis was associated with increased p53 expression. Moreover, silibinin enhanced UVB-induced apoptosis in MCF-7 cells.

Conclusion

Silibinin induced a loss of cell viability and apoptotic cell death in MCF-7 cells. Furthermore, the combination of silibinin and UVB resulted in an additive effect on apoptosis in MCF-7 cells. These results suggest that silibinin might be an important supplemental agent for treating patients with breast cancer.     

Intrinsic apoptotic pathway and G2/M cell cycle arrest involved in tubeimoside Ⅰ-induced EC109 cell death

Objective

Squamous esophageal carcinoma is highly prevalent in developing countries, especially in China. Tu Bei Mu (TBM), a traditional folk medicine, has been used to treat esophageal squamous cell carcinoma (ESCC) for a long term. tubeimoside I (TBMS1) is the main component of TBM, exhibiting great anticancer potential. In this study, we investigated the mechanism of TBMS1 cytotoxic effect on EC109 cells.

Methods

Comparative nuclear proteomic approach was applied in the current study and we identified several altered protein spots. Further biochemical studies were carried out to detect the mitochondrial membrane potential, cell cycle and corresponding proteins’ expression and location.

Results

Subcellular proteomic study in the nucleus from EC109 cells revealed that altered proteins were associated with mitochondrial function and cell proliferation. Further biochemical studies showed that TBMS1-induced molecular events were related to mitochondria-induced intrinsic apoptosis and P21-cyclin B1/cdc2 complex-related G2/M cell cycle arrest.

Conclusions

Considering the conventional application of TBM in esophageal cancer, TBMS1 therefore may have a great potential as a chemotherapeutic drug candidate for ESCC.Key Words: Anticancer drug, G2/M cell cycle arrest, intrinsic apoptosis, subcellular proteomics and tubeimoside I (TBMS1)     

DNA mismatch repair proficiency executing 5-fluorouracil cytotoxicity in colorectal cancer cells

Background

5-fluorouracil (5-FU)-based chemotherapy is the standard treatment for advanced stage colorectal cancer (CRC) patients. Several groups including ours have reported that stage II-III colorectal cancer patients whose tumors retain DNA Mismatch repair (MMR) function derive a benefit from 5-FU, but patients with tumors that lost MMR function do not. Although, MMR recognition of 5-FU incorporated in DNA has been demonstrated biochemically, it has not been demonstrated within cells to execute 5-FU cytotoxicity.

Aim

To establish an efficient construction model for 5-FU within DNA and demonstrate that 5-FU incorporated into DNA can trigger cellular cytotoxicity executed by the DNA MMR system.

Methods

We constructed a 5FdU-containing heteroduplex plasmid (5FdU plasmid) and 5FdU-containing linear ds-DNA (5FdU linear DNA), and transfected these into MMR-proficient, hMLH1^-/- and hMSH6^-/- cells. We observed cell growth characteristics of both transfectants for 5-FU-induced cytotoxicity.

Results

MMR-proficient cells transfected with the 5FdU plasmid but not the 5FdU linear DNA showed reduced cell proliferation by MTS and clonogenic assays, and demonstrated cell morphological change consistent with apoptosis. In MMR-deficient cells, neither the 5FdU plasmid nor 5FdU linear DNA induced cell growth or morphological changes different from controls.

Conclusion

5FdU as heteroduplex DNA in plasmid but not linear form triggered cytotoxicity in a MMR-dependent manner. Thus 5-FU incorporated into DNA, separated from its effects on RNA, can be recognized by DNA MMR to trigger cell death.Key words: 5-FU, colorectal cancer, mismatch repair system, heteroduplex plasmid     

Role of endoplasmic reticulum stress induction by the plant toxin,persin, in overcoming resistance to the apoptotic effects of tamoxifen in human breast cancer cells

Background:

Persin is a plant toxin that displays synergistic cytotoxicity with tamoxifen in human breast cancer cell lines. Here, we examined the ability of persin to circumvent tamoxifen resistance and delineated the intracellular signalling pathways involved.

Methods:

The induction of apoptosis in tamoxifen-resistant and -sensitive breast cancer cells was measured by flow cytometry following treatment with persin±tamoxifen. Markers of endoplasmic reticulum stress (ERS) were analysed following treatment, and their causal role in mediating persin-induced apoptosis was determined using chemical inhibitors and RNA interference.

Results:

Cells that were resistant to an apoptotic concentration of tamoxifen maintained an apoptotic response to persin. Persin-induced apoptosis was associated with an increase in markers of ERS, that is, CHOP expression and XBP-1 splicing and was decreased by CHOP siRNA. The CASP-4 inhibitor Z-YVAD-FMK markedly inhibited persin-induced apoptosis in both tamoxifen-sensitive and -resistant cells.

Conclusion:

The cytotoxic effects of persin are CASP-4 dependent and mediated by CHOP-dependent and -independent ERS signalling cascades. Increased ERS signalling contributes to persin-induced reversal of tamoxifen resistance.