The effect of cisplatin-low-level laser therapy on cell viability and death of LNCaP prostate cancer cell line.

Prostate cancer, as a common male cancer, is a serious threat to men's health. In spite of extreme developments for increasing survival rate, there are still limitations about common treatment options such as surgical procedures, radiotherapy, and chemotherapy. We hypothesized that combination of two treatments would bring better clinical outcomes. Therefore, the aim of this study was to determine the effect of conjugated cisplatin and low-level laser treatment (LLLT) on the viability of LNCaP prostate cancer cell line. LNCaP cells were harvested in DMEM containing 10% FBS and 1% antibiotic. Confluent cells were treated with different concentrations of cisplatin and different wavelengths of low-level laser (LLL) alone and in combination. The relative IC50 and cell viability was evaluated using MTT assay. Analysis of lipid peroxidation rate was performed using lipid peroxidation assay kit. LDH activity was also carried out on the treated and control cells using LDH cytotoxicity assay kit. Our results showed that combination of cisplatin and LLLT could effectively decrease cisplatin-induced cytotoxicity as well as LNCaP cell viability. Cisplatin-LLLT combination led to a significant increase in the MDA content as the product of membrane lipid peroxidation. Analyzing the LDH activity under the effect of cisplatin-LLL combined treatment showed a remarkable increase in the enzyme activity. We conclude that applying the cisplatin-LLL combination therapy is promising as an effective anti-cancer treatment. This novel combination has a potential to attenuate adverse side effects of earlier monotherapy strategies.

[Establishment of enzalutamide-resistant human prostate cancer cell lines and screening of lncRNA and mRNA expression profiles].

OBJECTIVE: To establish enzalutamide-resistant human prostate cancer cell lines and screen out the lncRNA and mRNA expression profiles associated with enzalutamide resistance. METHODS: Human prostate cancer cell lines LNCAP and C4-2B were cultured with 10 µmol/L enzalutamide for 6 months in vitro for the establishment of enzalutamide-resistant subclones LNCAP-ENZA and C4-2B-ENZA. The IC50 value and enzalutamide resistance index of each cell line were examined by MTT assay, the expressions of enzalutamide-related genes FL-AR, AR-V7 and HnRNPA1 were determined by Western blot, and the lncRNA and mRNA differential expressions of C4-2B and C4-2B-ENZA were detected by high-throughout lncRNA microarray. RESULTS: Compared with LNCAP and C4-2B, the IC50 values of enzalutamide-resistant subclones LNCAP-ENZA (60.83 µmol/L) and C4-2B-ENZA (88.32 µmol/L) were increased significantly (P < 0.05) and the enzalutamide-resistance indexes of the LNCAP-ENZA and C4-2B-ENZA cells were 4.94 and 4.67, respectively. The expressions of AR-V7 and HnRNPA1 were markedly up-regulated in the LNCAP-ENZA and C4-2B-ENZA cells as compared with those in the LNCAP and C4-2B cells, but that of FL-AR showed no significant change. A total of 1 440 lncRNAs and 1 236 mRNAs were identified as differentially expressed in the C4-2B-ENZA cells. CONCLUSIONS: Enzalutamide -resistant human prostate cancer cell subclones LNCAP-ENZA and C4-2B-ENZA were successfully established and enzalutamide resistance-associated lncRNA and mRNA were identified, which may provide some molecular evidence for the management of enzalutamide-resistant human prostate cancer.

Establishment of enzalutamide-resistant human prostate cancer cell lines and screening of lncRNA and mRNA expression profiles / 中华男科学杂志

Objective@#To establish enzalutamide-resistant human prostate cancer cell lines and screen out the lncRNA and mRNA expression profiles associated with enzalutamide resistance.@*METHODS@#Human prostate cancer cell lines LNCAP and C4-2B were cultured with 10 μmol/L enzalutamide for 6 months in vitro for the establishment of enzalutamide-resistant subclones LNCAP-ENZA and C4-2B-ENZA. The IC50 value and enzalutamide resistance index of each cell line were examined by MTT assay, the expressions of enzalutamide-related genes FL-AR, AR-V7 and HnRNPA1 were determined by Western blot, and the lncRNA and mRNA differential expressions of C4-2B and C4-2B-ENZA were detected by high-throughout lncRNA microarray.@*RESULTS@#Compared with LNCAP and C4-2B, the IC50 values of enzalutamide-resistant subclones LNCAP-ENZA (60.83 μmol/L) and C4-2B-ENZA (88.32 μmol/L) were increased significantly (P < 0.05) and the enzalutamide-resistance indexes of the LNCAP-ENZA and C4-2B-ENZA cells were 4.94 and 4.67, respectively. The expressions of AR-V7 and HnRNPA1 were markedly up-regulated in the LNCAP-ENZA and C4-2B-ENZA cells as compared with those in the LNCAP and C4-2B cells, but that of FL-AR showed no significant change. A total of 1 440 lncRNAs and 1 236 mRNAs were identified as differentially expressed in the C4-2B-ENZA cells.@*CONCLUSIONS@#Enzalutamide -resistant human prostate cancer cell subclones LNCAP-ENZA and C4-2B-ENZA were successfully established and enzalutamide resistance-associated lncRNA and mRNA were identified, which may provide some molecular evidence for the management of enzalutamide-resistant human prostate cancer.

Synthesis of Methotrexate Loaded Chitosan Nanoparticles and in vitro Evaluation of the Potential in Treatment of Prostate Cancer.

The objective of the study was to investigate the cytotoxic and apoptotic effects of Methotrexate (MTX)-loaded chitosan (CS) on LNCaP prostate cancer cell line in vitro. For this purpose, CS nanoparticles (NPs) were synthesized through ionic gelation method and MTX was loaded into the carrier with encapsulation. SEM images of the CS NPs have revealed that they have size of about 85 nm in mono-disperse manner. Drug loading yield was found to be 95.7 % with 470 µg drug/mg NP loading capacity. In vitro drug release study showed that MTX was released in a controlled manner. Cell viability was detected by using trypan blue dye exclusion test and WST-1 cell proliferation assay was performed to show cytotoxic effects of the CS, the MTX and the MTX-loaded NP. IC50 values of CS, MTX and MTX-loaded NPs were assigned from the cell survival plot and were determined as 67.18 µM, 20.21 µM and 2.94 µM at the 72nd hour, respectively. As for apoptosis analysis results, following to MTX-loaded CS treatment of LNCAP cells, apoptotic cell percent was detected as 39.3 % at the 72nd hour, that is, MTX-loaded CS induces 1.85-fold increase in apoptotic cell percent in comparison with that of MTX- induced apoptosis.

[Expression of pituitary tumor-transforming gene 1 during the development of androgen-independent prostate cancer].

OBJECTIVE: To explore the expression of pituitary tumor transforming gene 1 (PTTG1) during the transformation of prostate cancer from androgen-dependent (ADPC) to androgen-independent (AIPC). METHODS: We established an AIPC cell model LNCaP-AI by culturing the androgen-dependent LNCaP cell line in the hormone-deprived medium for over 3 months. The cell model was verified and the PTTG1 expression in the LNCaP cells was detected by Western blot and RT-PCR during hormone deprivation. RESULTS: The AIPC cell model LNCaP-AI was successfully established. The PTTG1 expression was gradually increased in the LNCaP cells with the prolonged time of hormone deprivation and the expressions of matrix metalloproteinases MMP-2 and -9 were elevated at the same time. CONCLUSIONS: The expression of PTTG1 is increased gradually in AIPC, which may be a target of gene therapy for advanced prostate cancer.

Expression of pituitary tumor-transforming gene 1 during the development of androgen-independent prostate cancer / 中华男科学杂志

<p><b>Objective</b>To explore the expression of pituitary tumor transforming gene 1 (PTTG1) during the transformation of prostate cancer from androgen-dependent (ADPC) to androgen-independent (AIPC).</p><p><b>METHODS</b>We established an AIPC cell model LNCaP-AI by culturing the androgen-dependent LNCaP cell line in the hormone-deprived medium for over 3 months. The cell model was verified and the PTTG1 expression in the LNCaP cells was detected by Western blot and RT-PCR during hormone deprivation.</p><p><b>RESULTS</b>The AIPC cell model LNCaP-AI was successfully established. The PTTG1 expression was gradually increased in the LNCaP cells with the prolonged time of hormone deprivation and the expressions of matrix metalloproteinases MMP-2 and -9 were elevated at the same time.</p><p><b>CONCLUSIONS</b>The expression of PTTG1 is increased gradually in AIPC, which may be a target of gene therapy for advanced prostate cancer.</p>

Study of NGEP expression in androgen sensitive prostate cancer cells: A potential target for immunotherapy.

BACKGROUND: Prostate cancer is one of the leading causes of cancer deaths among men. New gene expressed in prostate (NGEP), is a prostate-specific gene expressed only in normal prostate and prostate cancer tissue. Because of its selective expression in prostate cancer cell surface, NGEP is a potential immunotherapeutic target. To target the NGEP in prostate cancer, it is essential to investigate its expression in prostate cancer cells. METHODS: In the present study, we investigated NGEP expression in LNCaP and DU145 cells by real time and RT-PCR, flow cytometric and immunocytochemical analyses. RESULTS: Real time and RT-PCR analyses of NGEP expression showed that NGEP was expressed in the LNCaP cells but not in DU145 cells. The detection of NGEP protein by flow cytometric and immunocytochemistry analyses indicated that NGEP protein was weakly expressed only in LNCaP cell membrane. CONCLUSION: Our results demonstrate that LNCaP cell line is more suitable than DU145 for NGEP expression studies; however, its low-level expression is a limiting issue. NGEP expression may be increased by androgen supplementation of LNCaP cell culture medium.

Therapeutic Effect of Epigallocatechin-3-gallate (EGCG) and Silibinin on ATM Dynamics in Prostate Cancer Cell Line LNCaP.

BACKGROUND: Epigallocatechin-3-gallate (EGCG) is a major ingredient of green tea (GT) and silibinin (SB), the active component of Silymarin presumably hold a potential to prevent pathogenomics. Prostate cancer exacerbation is triggered by fusion transcripts formed because of genomic instability induced by juxtapositioning of two genes. This chimeric transcript is implicated in androgen dependent and independent prostate cancer. Tremendous work is done on the characterization of the mediators involved in the disease refractoriness, yet no study has addressed clinical management of these prostate fusion transcripts impressively. METHODS: An abolished ATM dynamics challenges integrity of DNA. In agreement with this assumption, ATM and DNA-PK were impaired in LNCaP cell line to confirm a tight interaction of these mediators with the expression profile of TMPRSS2-ERG. Abolished ATM enhanced the expression of the fusion transcript. Similarly blunting of DNA-PK downregulated the expression of the fusion transcript giving a notion that DNA-PK is involved in the chromosomal translocation. LNCaP cell lines were analyzed for the effect of EGCG and SB on the expression profile of TMPRSS2-ERG. RESULTS: In this particular unprecedented study, treatment of the LNCaP cell line with EGCG and Silibilin recapitulated ATM expression and activity and downregulated the fusion transcript appearance. CONCLUSIONS: These results underscore the therapeutic effect of EGCG and SB in mitigating the exacerbation of the disease with reference to the fusion transcripts.

Establishment of androgen-independent human prostate cancer line LNCaP by gradual deprivation of hormone / 第二军医大学学报

Objective: To establish and identify androgen-independent human prostate cancer cell line LNCap by culturing LNCaP cells with gradual deprivation of hormone. Methods: LNCaP cells were cultured in the medium with gradual deprivation of hormone (treated by active carbon to simulate androgen deprivation) for 10 days; and then the cells were cultured with complete deprivation of androgen for 3 months till the cell entered the rapid proliferation phase again. The cell growth and expression of PSA and androgen were examined by CCK-8, immunfluorescence and RT-PCR methods. Results: LNCaP cells grew slowly after deprivation of hormone and took on a neuroendocrine phenotype and cluster growth pattern. After 3 months' non-androgen culture,the cells regained original morphology and growth. CCK-8 indicated that LNCaP cells could grow in non-androgen condition; immunofluorescence assay indicated that LNCaP-AI cells could regain PSA-secreting activity in non-androgen condition; and RT-PCR suggested that androgen was highly expressed in LNCaP-AI cells. Conclusion: Androgen-independent LNCaP cell line can be established by culturing with gradual deprivation of hormone for 3 months.

Molecular Cloning and Characterization of a Putative Promoter Region of mPC-1 Gene Homologous to hPC-1 / 中国生物化学与分子生物学报

To identify the regulatory region that are responsible for the expression of mPC-1, we have isolated and characterized the mPC-1 gene promoter. Sequence analysis of the mPC-1 5' -flanking region and a series of truncated constructs were performed, which were transiently transfected into the prostate cancer cell lines and non-prostate cancer cell lines and analyzed through Dual-luciferase reporter assay system. The relative activity of mPC-1 gene promoter was by far higher than pGL3-control containing SV40 promoter and enhancer and p61-PSA containing hPSA 6 kb promoter in AR (androgen receptor, AR ) -positive prostate cancer cell lines. The region from 599 bp to 449 bp of mPC-1 promoter might contain a negative regulatory element. The expression of mPC-1 1.1 kb fragment is mainly restricted into prostate cancer cell lines. The relative activity of mPC-1 1.1 kb 5'-flanking region was regulated by androgen. The results demonstrated that the 1.1 kb fragment of mPC-1 5' -flanking region was relatively strong and prostate cancer cell specific promoter region.The 1.1 kb promoter of mPC-1 gene might be well suited to prostate cancer gene therapy if the promoter was properly modified.

Proteomic Analysis of a Human Prostate Cancer Cell Line after Incubation with a Novel Somatostatin14 Derivative.

BACKGROUND: Derivatives of somatostatin (sms) are sometimes used in the treatment of hormone-refractory prostatic cancer, in spite of modest results in controlled clinical studies. The optimal use in this context remains to be determined. The human prostatic cancer cell line LNCaP has been used in several previous proteomic analysis studies, which confirmed that sms indeed can affect the protein expression in this cell line. Proteomic analysis is an important tool to increase the understanding of how sms affects the protein expression of the tumor cell. MATERIALS AND METHODS: In this in vitro study, a new sms14 derivative, smsdx, a conjugate between sms14 and dextran, was incubated with an LNCaP cell culture. Sms14 was used as the positive control. Proteomic analysis, using rapid mini two-dimensional electrophoresis, was performed to determine the effects on protein expression. RESULTS: Marked quantitative differences were observed in the protein expression profiles in smsdx-treated LNCaP cells compared to negative control cells (untreated cells). Sets of 63 (yet unidentified) protein spots were differentially expressed. The difference was statistically significant (Mann-Whitney analysis). The 63 dataset was used to accurately discriminate control cells from smsdx-treated cells using hierarchical cluster analysis. Both similarities and differences in protein expression were observed between smsdx- and sms14-treated cells. CONCLUSION: Smsdx is a new sms14 derivative with long in vivo half-life and pan receptor affinity. Sms14-like effects on the protein expression of LNCaP cells seem to be preserved in the construct. The results convey new information about this potentially useful compound. Further studies are now in progress to identify the affected proteins.