VEGF receptor antisense therapy inhibits angiogenesis and peritoneal dissemination of human gastric cancer in nude mice

The efficacy of a phosphorothioate antisense oligonucleotide (ASO) for KDR/Flk-1 (KDR/Flk-1-ASO), an endothelial cell-specific vascular endothelial growth factor (VEGF) receptor, was investigated on the peritoneal dissemination and angiogenesis of a human gastric cancer cell line in nude mice. Green fluorescent protein (GFP)-transduced NUGC-4 (NUGC-4-GFP) human gastric cancer cells were implanted into the peritoneal cavity of nude mice. KDR/Flk-1-ASO, -SO, or phosphate-buffered saline was administrated from days 7 to 14, 200 microg/mouse, once a day. The mice were sacrificed on day 28. Disseminated peritoneal tumor nodules expressing GFP were visualized by fluorescence microscopy. KDR/Flk-1-ASO significantly decreased the extent of peritoneal dissemination of the tumors. The number of cells undergoing apoptosis was significantly increased in the KDR/Flk-1-ASO-treated tumors. Microvessel density was significantly reduced in the KDR/Flk-1-ASO-treated tumor nodules. The KDR/Flk-1 antisense strategy, therefore, decreases tumor dissemination apparently by inhibiting angiogenesis.     

Inhibition of nuclear factor-kappaB suppresses peritoneal dissemination of gastric cancer by blocking cancer cell adhesion

Currently, patients with peritoneal dissemination of gastric cancer must accept a poor prognosis because there is no standard effective therapy. To inhibit peritoneal dissemination it is important to inhibit interactions between extracellular matrices (ECM) and cell surface integrins, which are important for cancer cell adhesion. Although nuclear factor-kappa B (NF-κB) is involved in various processes in cancer progression, its involvement in the expression of integrins has not been elucidated. We used a novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), to study whether NF-κB blocks cancer cell adhesion via integrins in a gastric cancer dissemination model in mice and found that DHMEQ is a potent suppressor of cancer cell dissemination. Dehydroxymethylepoxyquinomicin suppressed the NF-κB activity of human gastric cancer cells NUGC-4 and 44As3Luc and blocked the adhesion of cancer cells to ECM when compared with the control. Dehydroxymethylepoxyquinomicin also inhibited expression of integrin (α2, α3, β1) in in vitro studies. In the in vivo model, we injected 44As3Luc cells pretreated with DHMEQ into the peritoneal cavity of mice and performed peritoneal lavage after the injection of cancer cells. Viable cancer cells in the peritoneal cavities were evaluated sequentially by in vivo imaging. In mice injected with DHMEQ-pretreated cells and lavaged, live cancer cells in the peritoneum were significantly reduced compared with the control, and these mice survived longer. These results indicate that DHMEQ could inhibit cancer cell adhesion to the peritoneum possibly by suppressing integrin expression. Nuclear factor-kappa B inhibition may be a new therapeutic option for suppressing postoperative cancer dissemination.     

Effective therapy for peritoneal dissemination in gastric cancer

Peritoneal dissemination is the most frequent cause of death from gastric cancer, accounting for death in 20% to 40% of patients. Preoperative intraperitoneal chemotherapy, peritonectomy, intraoperative chemohyperthermic perfusion, and early postoperative intraperitoneal chemotherapy are treatment modalities specifically designed to eliminate peritoneal dissemination and progression. Preoperative intraperitoneal chemotherapy is for containment of peritoneal free cancer cells, and also may facilitate complete eradication of visible peritoneal dissemination by peritonectomy. Further, complete cytoreduction can be achieved more often when peritonectomy is included in the surgical treatment of gastric cancer with peritoneal dissemination. Phase III data shows prolonged survival attributed to complete cytoreduction. Aggressive cytoreduction of peritoneal dissemination by peritonectomy can reduce residual tumor burden to micrometastases on the peritoneal surface that can be treated by intraoperative intraperitoneal chemotherapy and early postoperative intraperitoneal chemotherapy. Among all these modalities, surgical cytoreduction is probably the most important for survival benefit. If the surgical cytoreduction is visibly incomplete, prolonged survival cannot be expected, despite subsequent treatment. The surgeon's goal is to reduce the cancer cell burden to a microscopic level. Continued refinement of phase II studies is needed for maximal benefit and to standardize the technical and chemotherapeutic options of each modality.     

Prediction and treatment of peritoneal dissemination in gastric cancer

In advanced gastric cancer, the frequency of relapses such as metastasis to the peritoneum is high. For this reason, prognostic and treatment methods were studied. In 457 cases in which diagnostic cytology was utilized, 36 (61%) of the 59 cases in which dissemination had been macroscopically observed (P 1) were positive. Moreover, 13 cases of P 0 were also positive. The prognosis of the positive cases was worse, but there was not a significant statistical difference between the positive and negative cases. Chemotherapy has become the most common treatment because of the appearance of new anticancer drugs. TS-1 and paclitaxel were repeatedly administered in 10 cases, and the median survival time was 17 months. These drugs were effective even in carcinoma of the peritoneum, and an improvement in the prognosis can be expected. Surgery was performed in 23 cases due to stenosis of the digestive tract, and in 21 cases the patients were able to eat after surgery. The median postoperative survival time was 7 months, and surgery improved the prognosis. The improved sensitivity of diagnostic cytology and the standardization of chemotherapy and surgery warrant further study.     

Inhibition of DNA methylation by antisense oligonucleotide MG98 as cancer therapy

Results of preclinical studies and clinical phase I/II trials suggest that the antisense oligodeoxynucleotide and DNA methyltransferase inhibitor MG98 can safely and effectively lead to reactivation of methylation silence tumor suppressor genes. It is possible that daily or continuous dosing of MG98 might be more active and less toxic than intermittent dosing. Combination of MG98 with other agents having completely different mechanisms of action seems reasonable. One clinical trial now under way is evaluating the use of MG98 in combination with interferon-alpha in patients with advanced renal cell carcinoma (RCC). Because of the current preclinical and clinical evidence, further trials of MG98 as therapy for RCC would be of interest.     

Neoadjuvant chemotherapy for gastric cancer with peritoneal dissemination

An early detection and treatment of gastric cancer with peritoneal dissemination are rather difficult so that a clinical trial has been neglected. Therefore, we introduced a pre-operative peritoneal lavage diagnosis for gastric cancer patients with serosa-invaded tumors. Neoadjuvant chemotherapy was introduced to patients with positive cytology and with no non-curative factors except peritoneum. The neoadjuvant chemotherapy followed by surgery was done safely. The patients with the disappearance of CY and P factors due to neoadjuvant chemotherapy had a better prognosis than those with positive results of CY or R However, many of the patients with negative results from peritoneum eventually suffered a peritoneal recurrence. We started another protocol study with S-1 and an intra-peritoneal chemotherapy using docetaxel. The efficacy in the protocol result will be expected.     

Intraperitoneal chemotherapy in gastric cancer patients with peritoneal dissemination

Peritoneal dissemination is one of the non-curative factors in gastric cancer and colon cancer. Although many treatments have been conducted for peritoneal dissemination, no standard chemotherapy has yet been established. For sometime we had used continuous hyperthermic peritoneal perfusion (CHPP)for peritoneal dissemination in gastric cancer and colon cancer. CHPP has a marked survival benefit for scirrhous type gastric cancer patients without liver metastasis. Patients with prophylactic CHPP have significantly better prognoses than those without prophylactic CHPP, and therapeutic CHPP has a survival benefit for gastric cancer patients with slight to moderate peritoneal dissemination (P 1-2). But CHPP has no significant prognostic benefit for gastric cancer patients with severe peritoneal dissemination (P 3). Therefore, a new cancer treatment is needed for those patients. On the other hand, many kinds of anticancer agents, including cisplatin, via intraperitoneal (ip) administration have been tried thus far for peritoneal dissemination therapy. Especially, intraperitoneal taxane anticancer agent is very effective for the treatment and local control of severe peritoneal dissemination in gastric cancer. A phase I/II study of taxane anticancer agents via ip administration should be tried in gastric cancer patients with peritoneal dissemination.     

Neoadjuvant treatment of gastric cancer with peritoneal dissemination.

AIMS: To report our experience of neoadjuvant intraperitoneal and systemic chemotherapy (NIPS) for patients having a complete resection of the primary gastric cancer and peritoneal carcinomatosis (PC). PATIENTS AND METHODS: Patients with advanced peritoneal dissemination of primary gastric cancer had the placement of a peritoneal port system. For intraperitoneal chemotherapy, 40 mg of docetaxel and 150 mg of carboplatin were introduced in 1000 ml of saline on a weekly basis. Simultaneously, 100 mg/m2 of methotrexate and 600 mg/m2 of 5-fluorouracil were infused via a peripheral vein. A minimum of two cycles and up to six cycles of NIPS were used prior to cancer resection. At surgery a complete removal of the primary gastric cancer and the peritoneal implants by peritonectomy was attempted. RESULTS: Sixty-one patients were enrolled in the study. Thirty-nine had positive intraperitoneal cytology which reverted to negative cytology after treatment in 22. Thirty-eight showed a partial response. Thirty patients came to resection and 14 patients could be made disease-free. Median survival time of all patients was 14.4 months. Patients who received a complete resection had a median survival time of 20.4 months. Grade III/IV toxicities were not found after two courses of NIPS, but did develop in seven patients after more than three courses of NIPS. CONCLUSION: NIPS can downstage large volume peritoneal dissemination of gastric cancer. When combined with gastrectomy including peritonectomy a complete surgical resection was possible in one-quarter of the patients and resulted in a prolonged survival. This combined intraperitoneal and systemic chemotherapy for PC from gastric cancer is worthy of consideration for phase III clinical investigations.     

A novel experimental mouse model of peritoneal dissemination of human gastric cancer cells: different mechanisms in peritoneal dissemination and hematogenous metastasis.

We established a new cell line, AZ-P7a, with high peritoneal-metastatic potential in nude mice. AZ-P7a cells were derived from the human gastric carcinoma line AZ-521, which has low capacity for peritoneal dissemination. AZ-P7a cells developed peritoneal metastasis in 11 / 14 (78.6%) mice, whereas the parental AZ-521 cells developed metastasis in 2 / 6 (33.3%) mice. The metastatic foci in the peritoneum showed essentially the same histological appearance as those induced by parental cells. The tumorigenicity and the motile activity of AZ-P7a cells were stronger than those of the parental AZ-521 cells; in contrast, adhesion to the extracellular matrix and the production of vascular endothelial growth factor by AZ-P7a cells were decreased. In fluorescence-activated cell sorter (FACS) analysis, AZ-P7a cells expressed significantly greater levels of integrins alpha2, alpha3, alpha5, alpha6 and alphavbeta5, as compared with AZ-521 cells. However, alpha1, alpha4, alphavbeta3, hCD44H, hCD44v3, hCD44v6 and hCD44v10 were not expressed in either cell line. AZ-P7a cells developed no liver metastasis when administered by the intrasplenic injection method, though the highly liver metastatic cell line AZ-H5c showed the same rate of peritoneal dissemination as that exhibited by AZ-P7a cells after intraabdominal injection. These findings suggested that the mechanism of peritoneal dissemination differed from that of hematogenous metastasis. Moreover, the latter appears to be controlled by more complex mechanisms than the former. Thus, this cell line might be useful for investigating the mechanism of peritoneal dissemination of human gastric cancer.     

Treatment results of peritoneal dissemination from gastric cancer by neoadjuvant intraperitoneal-systemic chemotherapy

No standard treatment exists for peritoneal dissemination from gastric cancer. We reviewed our experience using a novel treatment consisting of peritonectomy and intraoperative chemo-hyperthermic peritoneal perfusion (CHPP). Records of all patients who underwent CHPP and cytoreductive surgery from 1992 to 2001 were reviewed. RESULTS: Data from 107 patients (average age, 52 years) were available. P3 dissemination was found in 72 patients, and 8 and 27 patients showed P1 or P2 dissemination, respectively. Peritoneal metastasis was synchronous in 75 and metachronous in 32 patients. All patients received CHPP after cytoreductive surgery. Peritonectomy was performed in 42 patients. Complete cytoreduction (CC-0) was achieved in 47 patients (44%). Peritonectomy, resulted in CC-0 in 69% (29/42), but CC-0 was achieved in 18 of 65 (28%) patients by ordinary surgical techniques. There were 23 postoperative complications (21%) after operation. The overall operative mortality was 2.8% (3/107). Median follow-up for the entire study group was 46 months. Seventeen patients (15%) were disease-free, and 90 patients were dead at the time of analysis. Eighty-seven deaths were related to progression of disease. The median survival of all patients was 16.2 months, with an actual 5-year survival of 6%. Median survival of CHPP plus ordinary cyoreduction was 12.0 months and that after CHPP and peritonectomy was 22.8 months. Completeness of cytoreduction and peritonectomy were significant prognostic factors on univariate analysis and 5-year survival rate was 27%. Lymph node status, grade of peritoneal dissemination (P1-2 vs P3), age (>60 years vs <60 years), tumor volume of dissemination (>2.5 cm vs <2.5 cm in diameter), and histologic type (differentiated vs. poorly differentiated type) did not affect survival. The cox proportional model demonstrated that completeness of cytoreduction was the strongest prognostic factor. Patients who had an incomplete resection had 2.8-fold higher risk of dying from disease than patients who underwent complete cytoreduction. The 5-year survival after complete cytoreduction was 12%, compared with 2% for incomplete resection. Four patients lived more than 5 years. Cytoreduction was incomplete in one 5-year survivor who showed complete response to CHPP. CONCLUSION: Complete cytoreduction using peritonectomy and CHPP may improve survival of patients with peritoneal dissemination from gastric cancer. This procedure is most appropriate for highly motivated patients who are committed to survive as long as possible.     

Treatment strategy for primary gastric cancer with peritoneal dissemination

Curative resection is considered to be a standard therapy for gastric cancer with localized peritoneal metastases. For tumors with diffuse dissemination, chemotherapy may play a major role, however, the benefits of reduction surgery and standard chemotherapy have not yet been clarified. Median survival time after reduction surgery was reported to be 4-13 months for patients diagnosed by surgery and/or CT and 5-6 months for chemotherapy for those diagnosed by CT alone. Reduction surgery has a high risk, with a morbidity of 12-44% and a mortality of 3-14%. Palliative surgery should be indicated for stenosis or bleeding due to primary tumors. 5-FU, MTX-5-FU, TS-1, paclitaxel, and their combination are candidates for practice and clinical trials. It is important to evaluate the severity of peritoneal dissemination by diagnostic laparoscopy or laparotomy for decision making.     

Modified pharmacokinetic modulating chemotherapy for progressive gastric cancer accompanied by peritoneal dissemination

Peritoneal dissemination is a major event in the development of gastric cancer. However, most patients with it have been excluded from clinical studies because they rarely have measurable lesions. We conducted an analysis to evaluate the efficacy and feasibility of modified pharmacokinetic modulating chemotherapy, for gastric cancer patients with peritoneal dissemination. Between May 2002 and April 2004, 10 patients were treated by modified pharmacokinetic modulating chemotherapy. This analysis was based on 10 consecutive chemotherapy-naive patients with confirmed peritoneal dissemination. This therapy regimen was repeated with a weekly schedule of MTX 100 mg/body, given as intraarterial infusion 1 h prior to a 24-hr infusion of 5-FU 500 mg/body. Simultaneously, enteric-coated tegafur/uracil (400 mg) was administered every day. The one-year overall survival rate was 50. 0%. The median survival time was 311 days. Grade 1 stomatitis and Grade 1/2 oral dryness were involved in 40% of the cases. No patient had to discontinue this therapy because of complications. Objective improvement of ascites was seen in all patients, and all patients could be treated at outpatient clinics. This regimen may be well-tolerated and of clinical benefit for patients with peritoneal dissemination of gastric cancer.     

Quantitative prognostic indicators of peritoneal dissemination of gastric cancer.

There are three classifications that describe the quantitative prognostic indicators of peritoneal dissemination for gastric cancer. The Japanese classification (P1, P2, and P3, Lyon classification, (stage I, II, stage III, and stage IV), and the Peritoneal Cancer Index (PCI). Carcinomatosis with limited extent (P1/ P2) corresponds to the PCI less than 13 and the stage I and II from Lyon classification. Carcinomatosis with large extent (P3) corresponds to PCI of 13 or larger and stage III and IV from Lyon classification. PCI enables one to describe the precise distribution of peritoneal dissemination. All three classifications correlate with prognosis. With regard to the surgical cytoreduction of the primary tumor and the peritoneal dissemination, Sugarbaker proposed the classification of completeness of cytoreduction (CCR). Patients with no macroscopic residual tumor had significantly better prognosis than those with residual disease. CCR is a valuable prognostic indicator after cytoreductive surgery.     

Mechanisms of the formation of the peritoneal dissemination in gastric cancer

To clarify the mechanisms of the formation of peritoneal dissemination, a new animal model by the i.p. inoculation of highly metastatic gastric cancer cell line MKN-45-P was developed. Peritoneal dissemination with bloody ascites was found in 100% of nude mice, injected 1x10(7) MKN-45-P cells in suspension into the peritoneal cavity. By a highly sensitive method for specific detection of metastasized human tumor cells in nude mice using polymerase chain reaction, a human beta-globin-related sequence in the DNA from various parts of the peritoneum was specifically amplified and detected by gel electrophoresis and by a specific oligonucleotide probe. Greater omentum showed a strong signal of the amplified fragments of human beta-globin gene from the 1st day and the signals gradually increased. The signals in the gonadal fat, mesentery and ovarium could be weakly detected on the Ist day, transiently decreased on the 3rd day, and then increased from the 7th day. In the diaphragm, and abdominal wall, signals could be detected from the 7th day. In contrast, small intestine and colon did not show any human beta-globin signals. In greater omentum and gonadal fat, cancer cells were selectively detected in the milky spots stained by activated carbon on the 3rd day. In the diaphragm, cancer cells adhered to the small pores termed stomata, and invaded into the subdiaphragmatic lymphatic lacunae connected with stomata. From the 3rd day, mesothelial cells of the abdominal cavity became round and separated, resulting in the exposure of the underlying connective tissue. MKN-45-P cells were found to adhere to the naked areas of the submesothelial connective tissue. From these results, we conclude that the major metastatic route of the peritoneum may be firstly through milky spots, secondly through the diaphragmatic stomata, and thirdly by the adhesion to the naked connective tissue exposed after shrinkage of the mesothelial cells. The third process may be related to the interaction between some adhesion molecules and their ligands.     

Effectiveness of continuous hyperthermic peritoneal perfusion for the peritoneal dissemination of gastric cancer

We investigated the effectiveness of continuous hyperthermic peritoneal perfusion (CHPP) for the peritoneal dissemination of gastric cancer. A total 124 patients with advanced gastric cancer were enrolled in this study. Prophylactic CHPP (P-CHPP) was performed in 45 patients who had macroscopic serosal invasion without peritoneal dissemination, and 79 patients without CHPP were a control group. Therapeutic CHPP (T-CHPP) was performed in 21 patients with peritoneal dissemination, and 52 patients without CHPP were a control group. There was no significant difference in 5 year survival between patients treated and not treated with P-CHPP. Univariate analysis showed that location of tumor, tumor diameter, and lymph node metastasis influenced prognosis, but there was no prognostic factor in the Cox proportional regression hazard model. There was no significant difference in 5-year survival between patients treated and not treated with T-CHPP. Univariate analysis showed that degree of peritoneal dissemination and adjuvant chemotherapy influenced prognosis, and the Cox proportional regression hazard model showed that the macroscopic types and degree of peritoneal dissemination affected prognosis. In the patients with CHPP, the incidences of respiratory failure and renal failure were each statistically greater than in the patients undergoing CHPP.     

Inhibition by rat C-erbB-2/neu antisense oligonucleotide of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats.

The effect of prolonged administration of a rat C-erbB-2/neu (C-erbB-2) antisense oligonucleotide on gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and on the labeling and apoptotic indices of gastric cancer was examined in Wistar rats After oral treatment with MNNG for 25 weeks, the rats received intraperitoneal injections of a C-erbB-2 antisense-liposome complex or a sense-liposome complex at a dose of 50 microgram oligonucleotide/kg body weight every other day until the end of the experiment in week 52. In week 52, the incidence of gastric cancers was significantly lover in rats treated with the C-erbB-2 antisense oligonucleotide than in rats treated with the sense oligonucleotide. Administration of the C-erbB-2 antisense oligonucleotide also significantly decreased the bromodeoxyuridine-labeling index and significantly increased the apoptotic index of gastric cancers. The mean cellular fluorescence of gastric antral cells in MNNG-treated rats was positively correlated with the dose of FITC-labeled C-erbB-2 antisense oligonucleotide. Our findings indicate that the antisense oligonucleotide inhibits gastric carcinogenesis through decreased cell proliferation and increased apoptosis induction and suggest that antisense strategies may provide new treatment for gastric cancer.