A Feasibility Study of Neoadjuvant XELOX Without Radiotherapy for Locally Advanced Lower Rectal Cancer.

AIM: This study was planned to evaluate the efficacy and safety of preoperative capecitabine and oxaliplatin (XELOX) without radiation in patients with locally advanced lower rectal cancer. PATIENTS AND METHODS: Patients with clinical stage II/III lower rectal cancer underwent three cycles of XELOX followed by radical surgery. The primary end-point was the R0 resection rate. RESULTS: Thirty-one patients were recruited between February 2012 and August 2014. The completion rate of neoadjuvant chemotherapy was 96.5% among the 29 patients who received it; the remaining two refused chemotherapy and underwent immediate surgery. Grade 3-4 adverse events occurred in nine patients (31%). All 29 patients who received chemotherapy underwent radical resection. The R0 resection rate was 96.5% among these 29 patients. Pathological complete responses were achieved in three patients (10.3%) and downstaging occurred in 13 (44.8%). CONCLUSION: This pilot study found that neoadjuvant XELOX for locally advanced lower rectal cancer is feasible and safe. This neoadjuvant treatment improved resection margin status.

Adenoviral therapy is more effective in gemcitabine-resistant pancreatic cancer than in gemcitabine-sensitive cells.

BACKGROUND: Although gemcitabine is the standard treatment for pancreatic cancer, this particular type of cancer develops rapidly and has intrinsic chemoresistance. Chemoresistance plays a critical role in tumor progression, invasion and migration. Nevertheless, the effect of adenoviral therapy on chemoresistant cancer cells has not been studied. In this study, we compared the efficacy of adenoviral therapy in parental and chemoresistant pancreatic cancer cells. MATERIALS AND METHODS: To establish gemcitabine-resistant cells, pancreatic cancer SUIT2 cells were exposed to increasing concentrations of gemcitabine. Both parental and chemoresistant cells were infected with adenoviruses expressing either green fluorescent protein (Ad-GFP) or the hepatocyte growth factor antagonist, NK4 (Ad-NK4). To investigate the transduction efficacy, GFP expression and NK4 concentrations were measured and an invasion assay was used to investigate the efficacy of the adenoviral therapy. RESULTS: The 50% inhibitory concentration of gemcitabine was <10 nM in the parental SUIT-2 cells, while it was >1 µM in gemcitabine-resistant cells. A large number of gemcitabine-resistant cells were GFP-positive compared with only a small number of parental cells (p<0.05). The NK4 expression level was significantly higher in gemcitabine-resistant cells than in parental cells (p<0.05). The supernatant from Ad-NK4-infected gemcitabine-resistant cells significantly inhibited the invasion of cancer cells compared with that from Ad-NK4-infected parental cells (p<0.05). CONCLUSION: Both the efficiency of transduction and the therapeutic efficacy of adenoviral therapy were higher in gemcitabine-resistant cells than in parental cells, suggesting that adenoviral gene therapy is more effective in patients with gemcitabine-resistant pancreatic cancer.

Tumor-stroma interactions reduce the efficacy of adenoviral therapy through the HGF-MET pathway.

Many preclinical studies have shown the potential of adenovirus-based cancer gene therapy. However, successful translation of these promising results into the clinic has not yet been achieved. Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant desmoplastic stroma, and tumor-stromal cell interactions play a critical role in tumor progression. Therefore, we hypothesized that tumor-stroma interactions reduce the efficacy of adenoviral therapy. We investigated the effect of fibroblasts on adenovirus-based gene therapy using SUIT-2 and PANC-1 pancreatic cancer cells cultured with or without fibroblast-conditioned culture supernatant then infected with Ad-LacZ. After 48 h, the cells were stained for ß-galactosidase. The results showed that the number of ß-galactosidase-positive cells was significantly reduced after culture with fibroblast-conditioned supernatant (P < 0.05). Because the hepatocyte growth factor (HGF)/MET pathway plays an important role in tumor-stroma interactions we next investigated the involvement of this pathway in tumor-stroma interactions leading to the decreased efficacy of adenoviral therapy. SUIT-2 cells were cultured with or without SU11274 (a MET inhibitor) and/or fibroblast-conditioned culture supernatant, then infected with Ad-GFP. After 48 h, GFP-positive cells were counted. The number of GFP-positive cells in cultures containing fibroblast-conditioned supernatant plus SU11274 was significantly greater than in cultures without SU11274. In conclusion, our results suggest that stromal cells in PDAC reduce the efficacy of adenoviral therapy through a mechanism involving the HGF/MET pathway. Control of such tumor-stroma interactions may lead to improvements in adenoviral gene therapy for PDAC.

Combination with low-dose gemcitabine and hTERT-promoter-dependent conditionally replicative adenovirus enhances cytotoxicity through their crosstalk mechanisms in pancreatic cancer.

To overcome the limited clinical efficacy of conditionally replicative adenoviruses (CRAds), we investigated the effects of combination therapy with gemcitabine (GEM) and the hTERT-promoter-dependent CRAd (hTERT-CRAd), Ad5/3hTERTE1. This combination therapy exhibited enhanced cytotoxic effects on pancreatic cancer both in vitro and in vivo. Furthermore, we revealed that this enhancement effect was due to the multiple bidirectional interactions between hTERT-CRAd and GEM. The GEM-sensitizing effect of E1 expression derived from hTERT-CRAd, and the enhancement effect by GEM on hTERT promoter activity which led to the increase of adenovirus E1 and viral infectivity. This combination therapy may be a promising therapeutic approach for pancreatic cancer.

Tumor-stromal interactions with direct cell contacts enhance proliferation of human pancreatic carcinoma cells.

Pancreatic ductal adenocarcinoma is often characterized by an abundant desmoplastic stroma that is partially induced by activated pancreatic stellate cells (PSCs). Indirect co-culture has often been used to investigate the effects of cancer-stromal interactions on the proliferation of cancer cells, but the effects of cell-cell adhesion and juxtacrine signaling between cancer and stromal cells cannot be evaluated using this method. This study aimed to establish a simplified direct co-culture system that could be used to quantify populations of cancer cells in co-culture with PSCs, and to evaluate the effects of direct cell contact on the proliferation of cancer cells. We established three green fluorescent protein (GFP)-expressing pancreatic cancer cell lines and were able to quantify them with high reliability and reproducibility, even when co-cultured directly with PSCs, using a color plate reader. We assessed the differential effects of direct and indirect co-culture with PSCs on the proliferation of cancer cells, and found that the proliferation of GFP-expressing pancreatic cancer cell lines was dramatically enhanced by direct co-culture with PSCs, compared with the indirect co-culture system. We also found that direct co-culture of cancer cells and PSCs activated the Notch signaling pathway in both cell types. Direct cell contact between cancer cells and PSCs plays an important role in the control of cancer cell proliferation, and is essential to the understanding of tumor-stromal interactions.

Up-regulation of integrin beta3 in radioresistant pancreatic cancer impairs adenovirus-mediated gene therapy.

Adenovirus-mediated gene therapy is a promising approach for the treatment of pancreatic cancer. We previously reported that radiation enhanced adenovirus-mediated gene expression in pancreatic cancer, suggesting that adenoviral gene therapy might be more effective in radioresistant pancreatic cancer cells. In the present study, we compared the transduction efficiency of adenovirus-delivered genes in radiosensitive and radioresistant cells, and investigated the underlying mechanisms. We used an adenovirus expressing the hepatocyte growth factor antagonist, NK4 (Ad-NK4), as a representative gene therapy. We established two radioresistant human pancreatic cancer cell lines using fractionated irradiation. Radiosensitive and radioresistant pancreatic cancer cells were infected with Ad-NK4, and NK4 levels in the cells were measured. In order to investigate the mechanisms responsible for the differences in the transduction efficiency between these cells, we measured expression of the genes mediating adenovirus infection and endocytosis. The results revealed that NK4 levels in radioresistant cells were significantly lower (P < 0.01) than those in radiosensitive cells, although there were no significant differences in adenovirus uptake between radiosensitive cells and radioresistant cells. Integrin beta3 was up-regulated and the Coxsackie virus and adenovirus receptor was down-regulated in radioresistant cells, and inhibition of integrin beta3 promoted adenovirus gene transfer. These results suggest that inhibition of integrin beta3 in radioresistant pancreatic cancer cells could enhance adenovirus-mediated gene therapy.

Chemotherapeutic agents potentiate adenoviral gene therapy for pancreatic cancer.

Adenovirus-mediated gene therapy combined with chemotherapeutic agents is expected to represent a new approach for treating pancreatic cancer. However, there have been no reports of definitive effects of chemotherapeutic agents on adenovirus-mediated gene therapies. In the present study, we investigated the effects of chemotherapeutic agents on the transduction efficiency of an adenovirus-based gene therapy. Adenovirus (Ad-NK4) expressing NK4, which acts as a hepatocyte growth factor antagonist, was used as a representative gene therapy. Pancreatic cancer cells infected with Ad-NK4 were treated with chemotherapeutic agents (5-fluorouracil [5FU], cisplatin or etoposide), and the NK4 levels in their culture media were measured. To examine the effects of chemotherapeutic agents in vivo, Ad-NK4 was administered to subcutaneous tumors in mice after treatment with the agents, and the tumor NK4 levels were measured. The NK4 levels in culture media from cells treated with 5FU, cisplatin and etoposide were 5.2-fold (P = 0.026), 6-fold (P < 0.001) and 4.3-fold (P < 0.001) higher than those of untreated cells, respectively. The chemotherapeutic agents also increased Ad-NK4 uptake. The NK4 levels in tumors treated with 5FU, cisplatin and etoposide were 5.4-fold (P = 0.006), 11.8-fold (P < 0.001) and 4.9-fold (P = 0.017) higher than those in untreated tumors, respectively. The present findings suggest that chemotherapeutic agents significantly improve the efficiency of adenovirus-mediated gene transfer in pancreatic cancer. Furthermore, they will contribute to decreases in the adenovirus doses required for gene transfer, thereby controlling the side-effects of adenovirus infection in normal tissues.

Midkine mRNA is overexpressed in pancreatic cancer.

PURPOSE: Midkine (MK) has been reported to be a possible molecular marker for the diagnosis of pancreatic cancer. We investigated the feasibility of quantitative analysis of MK mRNA by quantitative real-time RT-PCR (qRT-PCR) as a promising tool for the diagnosis of pancreatic cancer. RESULTS: We found that pancreatic cancer tissues expressed significantly higher levels of MK mRNA than intraductal pancreatic mucinous neoplasm (IPMN) and non-neoplastic pancreatic tissues (P < 0.05); in contrast, we did not find any differences in MK mRNA expression between IPMN and non-neoplastic pancreatic tissues. Additionally, we observed that poorly differentiated carcinoma samples expressed higher levels of MK mRNA than well-differentiated carcinoma samples, although a significant difference was not observed. CONCLUSIONS: The present data suggests that quantitative analysis of MK mRNA provides an objective and sensitive evaluation and may be a promising modality for the diagnosis of pancreatic cancer and the prediction of its prognosis.

LIM only 4 is overexpressed in late stage pancreas cancer.

BACKGROUND: LIM-only 4 (LMO4), a member of the LIM-only (LMO) subfamily of LIM domain-containing transcription factors, was initially reported to have an oncogenic role in breast cancer. We hypothesized that LMO4 may be related to pancreatic carcinogenesis as it is in breast carcinogenesis. If so, this could result in a better understanding of tumorigenesis in pancreatic cancer. METHODS: We measured LMO4 mRNA levels in cultured cells, pancreatic bulk tissues and microdissected target cells (normal ductal cells; pancreatic intraepithelial neoplasia-1B [PanIN-1B] cells; PanIN-2 cells; invasive ductal carcinoma [IDC] cells; intraductal papillary-mucinous adenoma [IPMA] cells; IPM borderline [IPMB] cells; and invasive and non-invasive IPM carcinoma [IPMC]) by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: 9 of 14 pancreatic cancer cell lines expressed higher levels of LMO4 mRNA than did the human pancreatic ductal epithelial cell line (HPDE). In bulk tissue samples, expression of LMO4 was higher in pancreatic carcinoma than in intraductal papillary-mucinous neoplasm (IPMN) or non-neoplastic pancreas (p < 0.0001 for both). We carried out microdissection-based analyses. IDC cells expressed significantly higher levels of LMO4 than did normal ductal epithelia or PanIN-1B cells (p < 0.001 for both) or PanIN-2 cells (p = 0.014). IPMC cells expressed significantly higher levels of LMO4 than did normal ductal epithelia (p < 0.001), IPMA (p < 0.001) and IPMB cells (p = 0.003). CONCLUSION: Pancreatic carcinomas (both IDC and IPMC) expressed significantly higher levels of LMO4 mRNA than did normal ductal epithelia, PanIN-1B, PanIN-2, IPMA and IPMB. These results suggested that LMO4 is overexpressed at late stages in carcinogenesis of pancreatic cancer.

Quantitative analysis of hTERT mRNA levels in cells microdissected from cytological specimens.

Clinicians frequently require cytopathological assessment of tumor samples for preoperative diagnosis, but in some specimens, diagnosis remains inconclusive after cytological examination. To date, several molecular markers, including human telomerase reverse transcriptase (hTERT), have been assessed for the ability to detect malignancy. However, analyses using whole cytological specimens are generally affected by contamination of untargeted cells. The present study investigated the feasibility of more sensitive examination by quantitative mRNA analysis of target cells microdissected from cytological specimens. Laser capture microdissection (LCM) was used to obtain target cells from cytological specimens. hTERT mRNA levels were then measured in target cells by quantitative real-time RT-PCR (qRT-PCR). The effect of RNA fragmentation on qRT-PCR was also assessed. Total RNA from cytological specimens was sometimes fragmented to a large degree. To avoid the effect of RNA fragmentation, gene specific priming and PCR primers generating short PCR products were used and no difference in delta Ct values between fragmented and non-fragmented RNA were found. hTERT mRNA levels were measured in cells microdissected from 33 cytological specimens. The levels of hTERT mRNA were significantly higher in malignant cases compared to those in non-malignant cases (P = 0.0003). The sensitivity was 96.2%, even when the specificities were 100%. High levels of hTERT mRNA were also found in three cases that were not diagnosed as malignant by cytological examination. Quantitative assessment of hTERT mRNA levels in cells microdissected from cytological specimens is a potential diagnostic tool to potentiate cytological examination in diagnosing malignancy.

Down-regulation of deoxycytidine kinase enhances acquired resistance to gemcitabine in pancreatic cancer.

BACKGROUND: The functional roles of deoxycytidine kinase (dCK) in acquired resistance to gemcitabine remain unknown in pancreatic cancer. Here, the functional involvement of dCK in gemcitabine-resistance of pancreatic cancer was investigated. MATERIALS AND METHODS: The levels of the dCK gene as well as other gemcitabine-related genes (hENT1, RRM1 and RRM2) were analyzed in gemcitabine-resistant pancreatic cancer cells (GR cells) using quantitative real-time reverse transcription polymerase chain reaction. The effects of inhibition of these genes on sensitivity to gemcitabine were evaluated. RESULTS: In GR cells, expression of dCK was significantly reduced compared with that of parental cells (p < 0.05). The dCK-targeting siRNA significantly reduced gemcitabine sensitivity (p < 0.01) without affecting cell proliferation. The RRM1- and RRM2-targeting siRNAs increased gemcitabine sensitivity (p < 0.05) and reduced cell proliferation even without gemcitabine treatment. The hENT-targeting siRNA did not affect gemcitabine sensitivity or cell proliferation. CONCLUSION: Down-regulation of dCK specifically enhanced acquired resistance to gemcitabine in pancreatic cancer cells without affecting their proliferation.

Radiation enhances adenoviral gene therapy in pancreatic cancer via activation of cytomegalovirus promoter and increased adenovirus uptake.

PURPOSE: Adenovirus-mediated gene therapy combined with radiation is expected to be a new approach to treat pancreatic cancer. However, there are no reports of definitive effects of radiation on adenovirus-mediated gene therapies. In the present study, we investigated the effect of radiation on the transduction efficiency of an adenovirus-based gene therapy. EXPERIMENTAL DESIGN: We used adenovirus expressing NK4 (Ad-NK4), an antagonist for hepatocyte growth factor, as a representative gene therapy. Pancreatic cancer cells preinfected with Ad-NK4 were irradiated, and NK4 levels in culture media of these cells were measured. We investigated cytomegalovirus (CMV) promoter activity and uptake of adenovirus in these cells. To examine the effect of radiation in vivo, Ad-NK4 was given to irradiated subcutaneous tumors in nude mice, and NK4 levels in tumors were measured. RESULTS: NK4 levels in culture media of irradiated cells were 4.5-fold (P < 0.01) higher than those of nonirradiated cells. Radiation enhanced activation of the CMV promoter and adenovirus uptake (P < 0.01), leading to increased levels of NK4. We found that activation of p38 mitogen-activated protein kinase and up-regulation of dynamin 2 may be involved in the radiation-induced activation of the CMV promoter and adenovirus uptake, respectively. NK4 levels in irradiated tumors were 5.8-fold (P = 0.017) higher than those in nonirradiated tumors. CONCLUSIONS: The present findings suggest that radiation significantly improves the efficiency of adenovirus-mediated gene transfer in pancreatic cancer and probably contributes to decreasing the dose of adenovirus required for gene transfer and controlling side effects of adenovirus infection in nonirradiated normal tissue.

Increased expression of ADAM 9 and ADAM 15 mRNA in pancreatic cancer.

BACKGROUND: A disintegrin and metalloproteases (ADAMs) comprise a multifunctional family of membrane-anchored proteins. ADAM 9 and ADAM 15 are involved in cell migration and invasion. Expression of ADAM 9 and ADAM 15 was reported to be altered in several types of cancer. MATERIALS AND METHODS: Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure the expression of ADAM 9 mRNA in bulk pancreatic tissues. Results showed no significant difference in the expression of ADAM 9 mRNA between pancreatic cancer and non-neoplastic pancreas. Primary cultured pancreatic fibroblasts also expressed ADAM 9 mRNA. Therefore, a laser microdissection and pressure catapulting technique was employed to isolate cancer cells from tumor tissues. The expression of ADAM 9 and ADAM 15 mRNA was measured in microdissected samples (cancer cells, n = 11; normal epithelial cells, n = 13 for ADAM 9; cancer cells, n = 9; normal epithelial cells, n = 9 for ADAM 15). RESULTS: Pancreatic cancer cells expressed significantly higher levels of ADAM 9 and ADAM 15 mRNA than did normal pancreatic epithelial cells (p = 0.016 for ADAM 9; p = 0.004 for ADAM 15). CONCLUSION: ADAM 9 and ADAM 15 are involved in pancreatic cancer. Microdissection-based analysis appears to be indispensable for the accurate analysis of the expression of certain ADAM family members in pancreatic cancer.

S100P is an early developmental marker of pancreatic carcinogenesis.

PURPOSE: Our goal was to clarify the involvement and clinical significance of S100P in pancreatic carcinogenesis. EXPERIMENTAL DESIGN: We examined S100P expression in 45 bulk pancreatic tissues; in microdissected cells, including invasive ductal carcinoma (IDC) cells (20 sections), pancreatic intraepithelial neoplasia (PanIN) cells (12 sections), intraductal papillary mucinous neoplasm (IPMN) cells (19 sections), and normal epithelial cells (11 sections); and in pancreatic juice samples from 99 patients with pancreatic diseases (32 cancer, 35 IPMN, and 32 chronic pancreatitis samples). We used quantitative real-time reverse transcription-PCR with gene-specific priming to measure S100P in these various types of samples. RESULTS: In bulk tissue analyses, pancreatic cancer and IPMN expressed significantly higher levels of S100P than did nonneoplastic pancreas (P<0.017 and P=0.0013, respectively). Microdissection analyses revealed that IPMN expressed significantly higher levels of S100P than did IDC (P<0.0001) and PanIN (P=0.0031), although S100P expression did not differ between IDC and PanIN (P=0.077). In pancreatic juice analyses, cancer and IPMN juice expressed significantly higher levels of S100P than did pancreatitis juice (both P<0.0001). Receiver operating characteristic curve analyses revealed that measurement of S100P in pancreatic juice was useful for discriminating neoplastic disease from chronic pancreatitis (area under the curve=0.837; 95% confidence interval, 0.749-0.903). CONCLUSION: S100P may be an early developmental marker of pancreatic carcinogenesis, and measurement of S100P in pancreatic juice may be useful for early detection of pancreatic cancer or screening of early pancreatic carcinogenesis.

Overexpression of c-met in the early stage of pancreatic carcinogenesis; altered expression is not sufficient for progression from chronic pancreatitis to pancreatic cancer.

AIM: To investigate c-met expression during early pancreatic carcinogenesis. METHODS: We used 46 bulk tissues and 36 micro-dissected samples, including normal pancreas, chronic pancreatitis, and pancreatic cancer, for quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: In bulk tissue analyses, pancreatic cancer tissues expressed significantly higher levels of c-met than did chronic pancreatitis and normal pancreas tissues. c-met levels did not differ between chronic pancreatitis and normal pancreas tissues. In microdissection-based analyses, c-met was expressed at higher levels in microdissected pancreatic cancer cells and pancreatitis-affected epithelial cells than in normal ductal epithelial cells (both, P < 0.01). Interestingly, pancreatitis-affected epithelial cells expressed levels of c-met similar to those of pancreatic cancer cells. CONCLUSION: Overexpression of c-met occurs during the early stage of pancreatic carcinogenesis, and a single alteration of c-met expression is not sufficient for progression of chronic pancreatitis-affected epithelial cells to pancreatic cancer cells.