Determining programmed cell death ligand 1 expression in circulating tumor cells of patients with clear cell renal cell carcinoma and its correlation with response to programmed cell death protein 1 inhibitors.

OBJECTIVE: There is a great interest in determining whether the expression of the programmed cell death ligand 1 is correlated with the efficacy of immune checkpoint inhibitors in patients with clear cell renal cell carcinoma; however, primary tumor biopsies can only provide limited information. Therefore, we explored the expression of programmed cell death ligand 1 on circulating tumor cells, which is a potential predictor of therapeutic response. METHODS: Circulating tumor cells were isolated from 20 clear cell renal cell carcinoma patients based on cell surface markers targeting clear cell renal cell carcinoma using IsoFlux device, followed by identification according to cell morphology and immunofluorescence studies. Programmed cell death ligand 1 expression status and clinical correlations were also analyzed. RESULTS: Before treatment with programmed cell death protein 1 inhibitors, circulating tumor cells were detected in all patients, ranging from 1 to 22 (median 7), with 75% (15/20) of the patients having programmed cell death ligand 1 + circulating tumor cells. Circulating tumor cell programmed cell death ligand 1 expression did not correlate with the immunohistochemical staining of programmed cell death ligand 1 in primary tumors. During treatment with programmed cell death protein 1 inhibitors, the disease control rate was much higher in the patients harboring programmed cell death ligand 1 + circulating tumor cells (73%, 11/15) than others (20%, 1/5). We also found that changes in total circulating tumor cell numbers and programmed cell death ligand 1 + circulating tumor cell counts correlated well with the disease outcome. CONCLUSION: We showed that the presence of programmed cell death ligand 1 + circulating tumor cells before programmed cell death protein 1 inhibition treatment could be a prognosis predictive factor and that the dynamic changes in circulating tumor cell numbers may be used to monitor the therapeutic response. Our study confirms the possibility of programmed cell death ligand 1 + circulating tumor cell detection in clear cell renal cell carcinoma patients' blood samples, which can potentially be used as an individualized immunotherapy molecular biomarker for real-time exploration.

CSC-3436 switched tamoxifen-induced autophagy to apoptosis through the inhibition of AMPK/mTOR pathway.

BACKGROUND: Triple-negative breast cancer (TNBC) lacks specific therapeutic target and limits to chemotherapy and is essential to develop novel therapeutic regimens. Increasing studies indicated that tamoxifen, a selective estrogen receptor modulators (SERMs), has anti-tumor therapeutic effect in estrogen receptor α (ERα)-negative tumor. Here, we determined whether autophagy was activated by tamoxifen in TNBC cells. Moreover, CSC-3436 displayed strong and selective growth inhibition on cancer cells. Next, we investigated the anti-proliferation effect of combination of CSC-3436 plus tamoxifen on cell death in TNBC cells. RESULTS: Our study found that tamoxifen induces autophagy in TNBC cells. Endoplasmic reticulum stress and AMPK/mTOR contributed tamoxifen-induced autophagy. Interestingly, in combination treatment with CSC-3436 enhanced the anti-proliferative effect of tamoxifen. We found that CSC-3436 switched tamoxifen-induced autophagy to apoptosis via cleavage of ATG-5. Moreover, AMPK/mTOR pathway may involve in CSC-3436 switched tamoxifen-induced autophagy to apoptosis. The combination of tamoxifen and CSC-3436 produced stronger tumor growth inhibition compared with CSC-3436 or tamoxifen alone treatments in vivo. CONCLUSION: These data indicated that CSC-3436 combined with tamoxifen may be a potential approach for treatment TNBC.

Emodin modulates epigenetic modifications and suppresses bladder carcinoma cell growth.

The deregulation of epigenetics was involved in early and subsequent carcinogenic events. Reversing cancer epigenetics to restore a normal epigenetic condition could be a rational approach for cancer treatment and specialized prevention. In the present study, we found that the expression levels of two epigenetic markers, histone H3K27 trimethylation (H3K27me3), was low but histone H3S10 phosphorylation (pH3Ser10) was high in human bladder cancer tissues, which showed opposite expression patterns in their normal counterparts. Thus, we investigated whether a natural product, emodin, has the ability to reverse these two epigenetic modifications and inhibit bladder cancer cell growth. Emodin significantly inhibited the cell growth of four bladder cancer cell lines in a dose- and time-dependent manner. Emodin treatment did not induce specific cell cycle arrest, but it altered epigenetic modifications. Emodin treatment resulted in the suppression of pH3Ser10 and increased H3K27me3, contributing to gene silencing in bladder cancer cells. Microarray analysis demonstrated that oncogenic genes including fatty acid binding protein 4 (FABP4) and fibroblast growth factor binding protein 1 (HBP17), RGS4, tissue inhibitor of metalloproteinase 3 (TIMP3), WNT5b, URB, and collagen, type VIII, alpha 1 (COL8A1) responsible for proliferation, survival, inflammation, and carcinogenesis were significantly repressed by emodin. The ChIP assays also showed that emodin increased H3K27me3 but decreased pH3Ser10 modifications on the promoters of repressed genes, which indicate that emodin reverses the cancer epigenetics towards normal epigenetic situations. In conclusion, our work demonstrates the significant anti-neoplastic activity of emodin on bladder cancer cells and elucidates the novel mechanisms of emodin-mediated epigenetic modulation of target genes. Our study warrants further investigation of emodin as an effective therapeutic or preventive agent for bladder cancer.

Calcified, minimally fat-contained angiomyolipoma clinically indistinguishable from a renal cell carcinoma.

BACKGROUND: Angiomyolipomas are benign tumors of the kidney. Typical angiomyolipomas are usually recognized by identifying fat components before any intervention. On the contrary, solid renal masses without evident fatty components but containing calcifications on the computed tomography scan are suspicious for malignancy. However, as in this rare case, rules of diagnostic imaging are of exceptions. CASE PRESENTATION: A 40-year-old man presented with left flank pain. The plain X-ray showed multiple coarse calcifications of 4.0x3.2 cm in diameter on the left upper quadrant abdomen. Computed tomography scan further revealed a solid renal mass and inside the mass there were calcifications. The size of the tumor was 5.6×5.5×6.3 cm. We performed a radical nephrectomy, and the histopathology showed a minimally fat-contained angiomyolipoma of multiple calcifications. The patient was free of recurrence or metastases after a follow-up period of 3 years. CONCLUSION: An angiomyolipoma containing calcification is rare. An angiomyolipoma with minimal fat concomitant with calcifications is an even rarer presentation. It is very difficult to differentiate a minimal-fat angiomyolipoma with calcifications from a renal cell carcinoma preoperatively. In such a circumstance, a well-planned partial nephrectomy may be optimal for the patient, regardless of the tumor size.

Circulating Tumor Cells Predict Response of Neoadjuvant Chemotherapy in Patients with Bladder Cancer: A Preliminary Study.

This study aimed to explore the existence of circulating tumor cells (CTCs) in patients with muscle-invasive bladder cancer (MIBC) and their predictive potential for response to neoadjuvant chemotherapy (NAC). From 33 blood samples of MIBC patients, CTCs were isolated by cell surface markers and enriched by the IsoFlux™ device, followed by morphological and immunofluorescent identification. CTCs were detected at baseline in all samples. Immunofluorescence confirmed the tumor origin. MIBC patients were stratified by NAC response into the disease control (DC) and progressive disease (PD) groups. In the DC group, the number of CTCs decreased significantly after four courses of NAC (p < 0.0001). CTC counts in 7.5 mL after four NAC cycles were highly correlated with postoperative pathological T stage (p < 0.0001). Our study demonstrated that CTCs might represent a valuable predictive marker for NAC response in MIBC. CTC detection in MIBC patients could allow early arrangement of radical cystectomy for NAC non-responders to prevent disease progression while receiving the NAC courses.

Detection of circulating tumor cells as therapeutic markers in patients with penile squamous cell carcinoma: A preliminary study.

BACKGROUND: This study aimed to investigate the presence of circulating tumor cells (CTCs) in patients with penile squamous cell carcinoma (PSCC). METHODS: CTCs were isolated from 14 patients with PSCC, 6 patients with balanoposthitis, and 6 healthy individuals. CTCs were enriched based on cell surface markers and filtered through the IsoFlux device, followed by identification according to cell morphology and immunofluorescence studies. RESULTS: CTCs were found in all PSCC blood samples but not in balanoposthitis samples and samples from healthy individuals. Immunofluorescence studies confirmed the tumor origin. When the patients with PSCC were stratified according to metastatic inguinal lymph node status, a statistically significant difference was observed in the number of detected CTCs. CONCLUSION: Our study showed that CTCs in PSCC may represent a valuable marker for differentiating PSCC from other tumors. Based on the correlation with some clinical parameters, CTC analysis is possibly relevant for noninvasive monitoring of disease progression and prognosis. The results also suggested a potential role of CTCs in preventing overtreatment, such as inguinal lymph node dissection.

Involvement of purinergic neurotransmission in ketamine induced bladder dysfunction.

PURPOSE: Long-term ketamine abuse in humans causes significant lower urinary tract symptoms. However, the etiology of ketamine associated cystitis is still not clear. We created a mouse model of ketamine induced lower urinary tract dysfunction to explore the pathogenesis of this condition. MATERIALS AND METHODS: Female C57BL/6 mice randomly distributed into control and ketamine groups received daily intraperitoneal injection of saline and ketamine (100 mg/kg), respectively. Cystometry was done in each group at 4, 8 and 16 weeks. After sacrifice the bladders were harvested for isometric muscle tension recording and immunohistochemical examination. RESULTS: After 8 weeks of treatment body weight growth was significantly decreased in ketamine treated mice. Cystometry revealed a significantly decreased intercontraction interval (mean±SEM 237±9 vs 360±20 seconds, p<0.001) and decreased bladder capacity (0.1±0.004 vs 0.13±0.006 ml, p<0.001) in ketamine vs saline injected mice. Increased adenosine triphosphate evoked detrusor contraction developed in the ketamine group. Immunohistochemical examination revealed increased P2X1 receptor expression in ketamine treated mouse bladders while M2 and M3 receptor expression was unchanged. CONCLUSIONS: At 8 weeks mice treated with ketamine showed increased voiding frequency and decreased bladder capacity, the same symptoms that develop in human ketamine abusers. Enhanced noncholinergic contractions and P2X1 receptor expression in the ketamine bladder indicate that dysregulation of purinergic neurotransmission may underlie detrusor overactivity in cases of ketamine induced bladder dysfunction.

Programmed Cell Death Ligand 1 Expression in Circulating Tumor Cells as a Predictor of Treatment Response in Patients with Urothelial Carcinoma.

Programmed cell death ligand 1 (PD-L1) inhibitors are commonly used in treating advanced-stage urothelial carcinoma (UC). Therefore, this study evaluated the relationship between PD-L1 expression in circulating tumor cells (CTCs) and treatment response to PD-L1 inhibitors using blood samples collected from patients with UC (n = 23). Subsequently, PD-L1 expression and its clinical correlation were analyzed. All patients had CTCs before PD-L1 inhibitory treatment, of which 15 had PD-L1-positive CTCs. However, PD-L1-positive expression in CTCs was not correlated with PD-L1 expression in tumor biopsy samples. Patients with PD-L1-positive CTCs had better disease control (DC) rates than those without PD-L1-positive CTCs. Moreover, changes in the proportion of PD-L1-positive CTCs were associated with disease outcomes. Furthermore, the PD-L1-positive CTC count in 9 of 11 patients who achieved DC had significantly decreased (p = 0.01). In four patients with progressive disease, this was higher or did not change. PD-L1-positive CTCs at baseline could be used as a biomarker to identify patients suitable for PD-L1 blockade therapy. Dynamic changes in PD-L1-positive CTCs during the course of treatment are predictive factors of immunotherapy response and prognostic factors of disease control. Hence, PD-L1-positive CTCs could be employed as a real-time molecular biomarker for individualized immunotherapy.

Transgenic expression of decoy receptor 3 protects islets from spontaneous and chemical-induced autoimmune destruction in nonobese diabetic mice.

Decoy receptor 3 (DCR3) halts both Fas ligand- and LIGHT-induced cell deaths, which are required for pancreatic beta cell damage in autoimmune diabetes. To directly investigate the therapeutic potential of DCR3 in preventing this disease, we generated transgenic nonobese diabetic mice, which overexpressed DCR3 in beta cells. Transgenic DCR3 protected mice from autoimmune and cyclophosphamide-induced diabetes in a dose-dependent manner and significantly reduced the severity of insulitis. Local expression of the transgene did not alter the diabetogenic properties of systemic lymphocytes or the development of T helper 1 or T regulatory cells. The transgenic islets had a higher transplantation success rate and survived for longer than wild-type islets. We have demonstrated for the first time that the immune-evasion function of DCR3 inhibits autoimmunity and that genetic manipulation of grafts may improve the success and survival of islet transplants.

Dual degradation of aurora A and B kinases by the histone deacetylase inhibitor LBH589 induces G2-M arrest and apoptosis of renal cancer cells.

PURPOSE: This study is aimed at investigating antineoplastic efficacy of histone deacetylase inhibitor (HDACI) LBH589 on renal cell carcinoma (RCC) and elucidating the novel molecular mechanisms involved in growth arrest and apoptosis by targeting the important nonhistone molecules. EXPERIMENTAL DESIGN: We analyzed the growth-inhibitory effect of LBH589 on RCC by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in vitro and antitumor efficacy by xenograft experiments in vivo. To verify the associated molecular mechanisms involved in LBH589-mediated cell death and cell cycle progression by Western blotting and fluorescence-activated cell sorting analysis. RESULTS: HDACI LBH589 induced degradation of both Aurora A and B kinases through a proteasome-mediated pathway by targeting HDAC3 and HDAC6. The dual degradation of Aurora A and B kinases mediated by LBH589 resulted in inducing G2-M arrest and apoptosis of renal cancer cell lines and our results also showed that LBH589 potently inhibited renal cancer cell growth in vitro and suppressed tumor formation in vivo. The Aurora A and B kinases and HDAC3 are overexpressed in the human RCC tumor tissues examined, which make them perfect targets for HDACI LBH589 treatment. CONCLUSIONS: Our in vitro and in vivo data showed that LBH589 has potent anticancer effect of renal cancer cells. LBH589 and other HDACI treatment resulted in inducing G2-M arrest and apoptosis of renal cancer cells through degradation of Aurora A and B kinases by inhibition of HDAC3 and HDAC6. The clinical efficacy of LBH589 in the treatment of patients with metastatic RCC, especially those with high Aurora kinase and HDAC expression, is worthy of further investigation.

The relationship between varicoceles and obesity in a young adult population.

To determine whether a relationship between obesity and varicocele occurrence exists, the prevalence and severity of varicoceles related to obesity were investigated in a general population of young males. A total of 1050 young males attending the Navy Recruit Training Center were evaluated from their physical screening examinations. All subjects underwent history taking and physical examinations to evaluate for the presence and severity of varicocele. The anthropometric indexes including body mass index (BMI), waist circumference (WC) and waist-to-hip ratio (WHR) were recorded. All subjects were categorized by quartiles according to each anthropometric index. Means were compared with the Student's t-test. Severity was compared by analysis of variance testing and frequency was analysed using the chi-square method. Statistical significance was considered at p <0.05. A total of 490 (46.67%) subjects had varicoceles. The means of BMI, WC and WHR of those without varicoceles was 23.99 +/- 3.82 kg/m(2), 83.20 +/- 9.97 cm and 0.85 +/- 0.05, respectively. These judged values were greater than those with varicoceles (22.02 +/- 3.18 kg/m(2), 79.19 +/- 9.01 cm and 0.83 +/- 0.05) (p < 0.001). In the univariate regression analysis, BMI, WC and WHR all had a significantly negative correlation with severity of varicocele (all p < 0.001). Analysis comparing varicocele frequency based on each grade per anthropometric index group was performed. The logistic regression revealed that the prevalence of grade II and III varicoceles showed a statistically inverse association with all three anthropometric indexes. The prevalence and severity of varicoceles inversely correlated with obesity. The present data support the explanation that obesity may result in a decreased nutcracker effect, which accounts for prevention of the renal vein compression by the adipose tissue.

Ketamine-associated bladder dysfunction.

OBJECTIVE: To assess the impact of ketamine abuse on genitourinary tract dysfunction. METHODS: Eleven patients with urinary tract symptoms and a history of ketamine abuse in recent years were studied. Urinalysis, urine culture, renal function tests, abdominal sonography and urodynamic studies were done. Bladder biopsies were carried out in selected cases. RESULTS: The most common complaints were lower urinary tract symptoms, including dysuria, frequency, urgency and gross hematuria. Urinalyses showed nonbacterial pyuria and were negative for tuberculosis. All biopsy specimens showed infiltrations of granulocytes (mostly eosinophils) and mast cells within the bladder tissue. Medications produced only slight clinical improvements. Intravesical instillation of hyaluronan solution was performed for some patients and a significant improvement of lower urinary tract symptoms was observed. CONCLUSIONS: Although the dosage and duration of ketamine abuse causing severe side-effects are still unclear, some patients develop irreversible histological changes in the urinary tract. Therefore, clinicians should be aware of the negative effects of ketamine abuse on genitourinary tract function.

Laparoscopic radical prostatectomy: initial experience of robotic surgery in Taiwan.

BACKGROUND: The incidence of prostate cancer is lower in Taiwan compared to Europe and North America. Only a few urologists have been trained to perform laparoscopic radical prostatectomies (LRP). The initial experience of robotic LRP in Taiwan is reported. PATIENTS AND METHODS: Twenty-four patients with localized prostate carcinomas underwent robotic LRP between November 2004 and December 2006. Data collection included basic demographics, prostate specific antigen (PSA), stages, Gleason scores, operative time, estimated blood loss (EBL), catheterization time, pathology, complications and return of continence. RESULTS: The mean operative time was 310 minutes, the mean EBL was 234 mL and the average urethral catheterization time was 7.1 days. Three patients had a total of three complications. Continence was 20.8% at one week, 59.1% at six weeks and 75% at three months postoperatively. CONCLUSION: Our initial experience with robotic LRP indicated that the procedure was safe and effective. Six case experiences were required to establish a standard operating procedure. Eighteen operations were needed to complete the learning curve and achieve stable results.

HAF mediates the evasive resistance of anti-angiogenesis TKI through disrupting HIF-1α and HIF-2α balance in renal cell carcinoma.

Anti-angiogenesis has emerged as a standard of care for metastatic renal cell carcinoma. However, long-lasting efficacy is seldom reached, and evasive resistance eventually occurs under anti-angiogenic tyrosine kinase inhibitor (TKI) therapy. To establish new therapeutic strategies, investigating the molecular mechanism of resistance is critically important. In our study, human umbilical vascular endothelial cells (HUVECs) were incubated with TKI treatment in conditioned medium derived from renal cancer cells (RCCs) to demonstrate cell viability. Quantitative real time PCR or Western blotting analysis detected the fluctuation of transcriptional factors HIF-1α and HIF-2α in RCCs under TKI treatment. We demonstrated the alteration of a specific cytokine produced from RCCs under normoxia or hypoxia incubation by utilizing a cytokine RT-PCR primer array. We found that the anti-angiogenic TKI sunitinib disrupted the balance between HIF-1α and HIF-2α in RCCs and led to a protective effect on HUVECs against sunitinib treatment when cultured with conditioned medium. Mechanistically, RCCs treated with sunitinib resulted in down-regulation of HIF-1α, but not HIF-2α, through reduction of both mRNA and protein levels. The down-regulation of HIF-1α by sunitinib occurred via hypoxia associated factor (HAF), which also enhanced HIF-2α transactivation activity to increase the production of pro-angiogenic factors and cytokines and promote HUVEC proliferation. This phenomenon was observed in ACHN and A498 cells, which express both HIF-1α and HIF-2α, but was not observed in 786-O cells, which express only HIF-2α. Our results illustrated that targeting both angiogenesis and hypoxia pathways might provide a resolution to dealing with the devastating effects of anti-angiogenesis resistance.

Limited Wegener's granulomatosis of the epididymis and testis.

A case is presented of Wegener's granulomatosis limited to the testis and epididymis, simultaneously, in a 69-year-old man. Orchiectomy was carried out through an inguinal incision under the presumptive diagnosis of a right testicular tumor. A hard, irregular mass occupied the upper testicle and a portion of the epididymal head was visualized. Histopathologic examination of the specimen showed granulomatous inflammation of the testis and epididymis with prominent angiocentric granulomata in the walls of arteries, veins and foci of fibrinoid necrosis, surrounded by palisading inflammatory cells with a few giant cells. The diagnosis of limited Wegener's granulomatosis was considered, although antineutrophil cytoplasmic antibody (c-ANCA) test was negative 2 weeks after orchiectomy. The patient showed an excellent response after local complete excision. He remains free of disease 18 months after orchiectomy.

Emphysematous epididymo-orchitis as a camouflage of prostate invasion secondary to rectum cancer: A case report.

INTRODUCTION: Emphysematous epididymo-orchitis is a rare cause of acute scrotum pain characterized by gas formation within the tissue. Diabetes mellitus and recto-seminal fistula secondary to sigmoid diverticulitis are generally accepted as being responsible for this disease. However, prostate invasion secondary to rectal cancer may be considered to be a newly identified pathogenetic mechanism. Herein, we report this rare case and illustrate the pathogenesis. CASE PRESENTATION: A 69-year-old man arrived at our emergency department presenting with sepsis and acute scrotal pain. Emphysematous epididymo-orchitis was diagnosed by scrotal sonography initially; however, advanced rectal cancer with prostate invasion was diagnosed by CT after a recurrent episode. An exploratory laparotomy with abdominoperineal resection and radical prostectomy were performed after neoadjuvant chemoradiotherapy. Histopathologic analysis confirmed the previous diagnosis. Emphysematous epididymo-orchitis caused by advanced rectal cancer is very rare, and our case is the first to be reported according to a literature search. Neoadjuvant chemoradiotherapy plus extended surgery can achieve a good oncological outcome. CONCLUSION: This case indicated that the very rare presentation as emphysematous epididymo-orchitis caused by locally advanced colorectal cancer.

The association of Id-1, MIF and GSTpi with acquired drug resistance in hormone independent prostate cancer cells.

Most patients with prostate cancer respond to androgen ablation therapy, but the tumor eventually progresses to an androgen-independent stage with multiple anti-cancer drug resistance. Novel therapeutic strategies for hormone independent prostate cancer need to be developed. The genes for Id-1, MIF and GSTpi were up-regulated in drug resistant cells of hormone independent prostate cancer cells using cDNA microarray gene expression analysis. In this study we aimed to investigate whether constitutive overexpression of these candidate genes in cells can affect cellular resistance to chemotherapeutic agents. The mRNA expression was determined by reverse transcriptase-polymerase chain reaction, and the Id-1, MIF and GSTpi protein contents in these cell lines were measured by Western blotting. Susceptibility of the cells to chemotherapeutic agents including doxorubicin, paclitaxel and cyclophosphamide was determined by microculture tetrazolium bromide assay. The analysis of apoptosis was assessed by annexin V assay. The cytotoxity assay results demonstrated that cells overexpressing the Id-1 gene increased in their resistance to doxorubicin, paclitaxel and cyclophosphamide. MIF expression can drive cells to increase in their resistance to paclitaxel, and GSTpi expression confers drug resistance to doxorubicin and cyclophosphamide. The induction of mdr1 gene expression was noted in up-regulation of MIF and GSTpi. Our findings suggest that overproduction of the Id-1, MIF and GSTpi proteins and coinduction of mdr-1 play an important role in the development of acquired drug resistance to various drugs of prostate cancer cells.

Novel Cancer Therapeutics with Allosteric Modulation of the Mitochondrial C-Raf-DAPK Complex by Raf Inhibitor Combination Therapy.

Mitochondria are the powerhouses of cells. Mitochondrial C-Raf is a potential cancer therapeutic target, as it regulates mitochondrial function and is localized to the mitochondria by its N-terminal domain. However, Raf inhibitor monotherapy can induce S338 phosphorylation of C-Raf (pC-Raf(S338)) and impede therapy. This study identified the interaction of C-Raf with S308 phosphorylated DAPK (pDAPK(S308)), which together became colocalized in the mitochondria to facilitate mitochondrial remodeling. Combined use of the Raf inhibitors sorafenib and GW5074 had synergistic anticancer effects in vitro and in vivo, but targeted mitochondrial function, rather than the canonical Raf signaling pathway. C-Raf depletion in knockout MEF(C-Raf-/-) or siRNA knockdown ACHN renal cancer cells abrogated the cytotoxicity of combination therapy. Crystal structure simulation showed that GW5074 bound to C-Raf and induced a C-Raf conformational change that enhanced sorafenib-binding affinity. In the presence of pDAPK(S308), this drug-target interaction compromised the mitochondrial targeting effect of the N-terminal domain of C-Raf, which induced two-hit damages to cancer cells. First, combination therapy facilitated pC-Raf(S338) and pDAPK(S308) translocation from mitochondria to cytoplasm, leading to mitochondrial dysfunction and reactive oxygen species (ROS) generation. Second, ROS facilitated PP2A-mediated dephosphorylation of pDAPK(S308) to DAPK. PP2A then dissociated from the C-Raf-DAPK complex and induced profound cancer cell death. Increased pDAPK(S308) modification was also observed in renal cancer tissues, which correlated with poor disease-free survival and poor overall survival in renal cancer patients. Besides mediating the anticancer effect, pDAPK(S308) may serve as a predictive biomarker for Raf inhibitors combination therapy, suggesting an ideal preclinical model that is worthy of clinical translation.

Treatment of bladder carcinomas using recombinant BCG DNA vaccines and electroporative gene immunotherapy.

Intravesical immunotherapy with live Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the treatment of choice for superficial bladder cancers. Nevertheless, a significant proportion of patients do not respond to this therapy, and adverse effects are common. Here, we report the cloning of recombinant mycobacterial DNA vaccines and demonstrate the ability of multicomponent and multisubunit DNA vaccines to enhance Th1-polarized cytokine-mediated responses as well as effector cell responses. Splenocytes from immunized groups of mice were restimulated in vitro and examined for cytotoxicity against murine bladder tumur (MBT-2) cells. We used four combined recombinant BCG DNA vaccines (poly-rBCG) for electroporative gene immunotherapy (EPGIT) in vivo, and found that tumor growth was significantly inhibited and mouse survival was prolonged. Increased immune cell infiltration and induction of apoptosis were noted after treatment with poly-rBCG alone, with the murine interleukin-12 (mIL-12) vaccine alone, and-most significantly-with the poly-rBCG+mIL-12 vaccine combination. Electroporation of poly-rBCG+mIL-12 resulted in complete tumor eradication in seven of eight mice (P<.01) within 28 days. Thus, EPGIT using multicomponent multisubunit BCG is highly effective in the treatment of bladder cancer. This approach presents new possibilities for the treatment of bladder cancer using recombinant BCG DNA vaccines.