Yuichi Kakudo

Synthesis and biological analysis of new curcumin analogues bearing an enhanced potential for the medicinal treatment of cancer

Curcumin (diferuloylmethane) is a dietary phytochemical with low toxicity that exhibits growth-suppressive activity against a variety of cancer cells and possesses certain chemopreventive properties. Curcumin has already been the subject of several clinical trials for use as a treatment in human cancers. Synthetic chemical modifications of curcumin have been studied intensively in an attempt to find a molecule with similar but enhanced properties of curcumin. In this study, a series of novel curcumin analogues were synthesized and screened for anticancer activity. New analogues that exhibit growth-suppressive activity 30 times that of curcumin and other commonly used anticancer drugs were identified. Structurally, the new analogues are symmetrical 1,5-diarylpentadienone whose aromatic rings possess an alkoxy substitution at each of the positions 3 and 5. Analysis of the effects of the analogues on the expression of cancer-related genes usually affected by curcumin indicated that some induced the down-regulation of β-catenin, Ki-ras, cyclin D1, c-Myc, and ErbB-2 at as low as one eighth the concentration at which curcumin normally has an effect. The analogues, however, exhibited neither harmful nor growth-suppressive effects on normal hepatocytes where oncogene products are not activated. They also exhibited no toxicities in vivo that they may provide effective alternative therapies for the prevention and treatment of some human cancers. [Mol Cancer Ther 2006;5(10):2563–71]

Lack of Correlation between p53-Dependent Transcriptional Activity and the Ability to Induce Apoptosis among 179 Mutant p53s

Tumor suppressor p53-dependent apoptosis is thought to be one of the most important tumor-suppressive functions in human tumorigenesis. However, whether the major mechanism underlying the p53-dependent apoptosis is transactivation dependent or independent remains unclear. Using 179 mutant p53s with diverse transcriptional activities for distinct p53-binding sequences in yeast, we evaluated both their sequence-specific transcriptional activities on six p53 target genes and their ability to induce apoptosis in Saos-2 cells. These mutant p53s also represented diversity in their ability to both transactivate target genes and induce apoptosis. We identified 17 mutant p53s with superior ability to induce apoptosis than wild-type p53 that tend to cluster at residues 121 or 290 to 292. There was no significant correlation between the two functional properties on any single target gene examined. Furthermore, the 17 mutant p53s were not classified in a specific cluster by hierarchical cluster analysis on their diverse transcriptional activities, indicating that these mutant p53s were not similar in the transcriptional activity of downstream genes. These results suggested that transactivation-dependent apoptosis does not always play a major role in p53-dependent apoptosis, indirectly supporting the importance role of the transactivation-independent mechanism.

Newly synthesized curcumin analog has improved potential to prevent colorectal carcinogenesis in vivo

Curcumin (diferuloylmethane) has chemopreventive and chemotherapeutic potentials against various types of cancers. We have developed a series of curcumin analogs to improve its low bioavailability by enhancing its potentials. The newly synthesized analog GO‐Y030 [(1E, 4E)‐1,5‐bis‐(3,5(‐bismethoxymethoxyphenyl) penta‐1,4‐dien‐3‐one] showed a 30‐fold greater growth suppression in vitro via similar molecular mechanisms to curcumin. The availability of this analog was examined by using a mouse model harboring the germ‐line mutation of Apc, Apc580D/+, in vivo. Apc580D/+ mice had a very limited survival time with an intestinal obstruction due to polyposis. The average tumor number in mice fed GO‐Y030 was reduced to 61.2% of those that were fed the basal diet (P < 0.05). Compared with Apc580D/+ mice fed the basal diet (median survival time = 166.5 days), a significantly prolonged lifespan (213 days) was observed in Apc580D/+ mice fed GO‐Y030. The chemopreventive effect with GO‐Y030 was improved, compared with curcumin (191 days). The survival benefit corresponded to the diminished intestinal tumor incidence in Apc580D/+ mice fed GO‐Y030. No adverse reactions were observed, judging from body weight or biochemical data concerning liver and renal damage. Degradation of accumulated β‐catenin with curcumin is one of the major mechanisms of chemoprevention in colorectal carcinogenesis. It was demonstrated that the number of β‐catenin‐positive adenoma cells in Apc580D/+ mice fed GO‐Y030 was reduced. (Cancer Sci 2009; 100: 956–960)

α-Catenin is essential in intestinal adenoma formation

A loss-of-function mutation in the APC gene initiates colorectal carcinogenesis. Although the molecular mechanism of tumor initiation is complex, several modifier genes have been identified using mouse models, including the ApcMin mouse. Among the familial adenomatous polyposis mouse lines carrying a truncation mutation at codon 580 in Apc (Apc580D), one line (line19-Apc580D/+) showed a remarkably reduced incidence of intestinal adenomas (<5% compared with other lines). Extensive genetic analysis identified a deletion in the α-catenin (Ctnna1) gene as the cause of this suppression. Notably, the suppression only occurred when the Ctnna1 deletion was in cis-configuration with the Apc580D mutation. In all adenomas generated in line19-Apc580D/+, somatic recombination between the Apc and Ctnna1 loci retained the wild-type Ctnna1 allele. These data strongly indicate that simultaneous inactivation of α-catenin and Apc during tumor initiation suppresses adenoma formation in line19-Apc580D/+, suggesting that α-catenin plays an essential role in the initiation of intestinal adenomas. Although accumulating evidence obtained from human colon tumors with invasive or metastatic potential has established a tumor-suppressive role for α-catenin in late-stage tumorigenesis, the role of α-catenin in the initiation of intestinal tumorigenesis is not well documented, especially compared with that of β-catenin. A mouse model used in this study focused on the early stage of tumor initiation and clearly indicated an essential role for α-catenin. Thus, α-catenin has dual roles in intestinal tumorigenesis, a supporting role in tumor initiation, and a suppressive role in tumor progression.

The screening of the second-site suppressor mutations of the common p53 mutants.

Second‐site suppressor (SSS) mutations in p53 found by random mutagenesis have shown to restore the inactivated function of some tumor‐derived p53. To screen novel SSS mutations against common mutant p53s, intragenic second‐site (SS) mutations were introduced into mutant p53 cDNA in a comprehensive manner by using a p53 missense mutation library. The resulting mutant p53s with background and SS mutations were assayed for their ability to restore the p53 transactivation function in both yeast and human cell systems. We identified 12 novel SSS mutations including H178Y against a common mutation G245S. Surprisingly, the G245S phenotype is rescued when coexpressed with p53 bearing the H178Y mutation. This result indicated that there is a possibility that intragenic suppressor mutations might restore the protein function in an intermolecular manner. The intermolecular mechanism may lead to novel strategies for restoring inactivated p53 function and tumor suppression in cancer treatment.

Dose escalation study of docetaxel and nedaplatin in patients with relapsed or refractory squamous cell carcinoma of the esophagus pretreated using cisplatin, 5-fluorouracil, and radiation

BackgroundDefinitive chemoradiation with cisplatin (CDDP) and 5-fluorouracil (5FU) has been playing an important role in the treatment of esophageal cancer, but some patients are not curable or have recurrent lesions. However, few chemotherapeutic regimens are available for such patients. Docetaxel and nedaplatin are active for esophageal cancer. We conducted a dose-escalation study of docetaxel and nedaplatin as second line-chemotherapy after definitive chemoradiation in patients with relapsed or refractory squamous cell carcinoma of the esophagus after chemoradiation.MethodsNedaplatin was administered on day 1 and docetaxel was administered on days 1 and 15, every 4 weeks. Dose escalation was based on the dose-limiting toxicity (DLT) observed during the first cycle.ResultsTwelve patients were enrolled. At a docetaxel dose of 30 mg/m2 and a nedaplatin dose of 80 mg/m2, one grade 4 neutropenia occurred and caused one treatment break longer than 2 weeks, but there were few DLTs. At doses of 35 and 80 mg/m2, respectively, two grade 4 neutropenias and one grade 2 thrombopenia occurred and caused three treatment breaks longer than 2 weeks. Therefore, the maximum tolerated dose was established at this dose level. Two grade 3 anorexias and one grade 3 nausea occurred, but other non-hematological toxicities were generally mild. Responses were seen in one-fourth of the 12 patients, including one complete remission.ConclusionThe recommended doses of docetaxel and nedaplatin were 30 and 80 mg/m2, respectively. This combination could be a potential second-line treatment for this target population.

Phase I/II study of sequential therapy with irinotecan and S-1 for metastatic colorectal cancer

Background:Both irinotecan (CPT-11) and S-1 are active against colorectal cancer; however, as S-1 is a prodrug of 5-fluorouracil (5-FU), 5-FU and its metabolites might inhibit the antitumour effect of CPT-11. Therefore, we designed a sequential combination, in which CPT-11 infusion was given on day 1 and S-1 was given orally at 80 mg m−2 per day on days 3–16 every 3 weeks.Methods:Twelve patients entered the phase I study, and the recommended doses were determined as a CPT-11 dose of 150 mg m−2 and an S-1 dose of 80 mg m−2.Results:In all, 36 patients entered the phase II study, of whom 4 and 16 had complete and partial responses. The overall response rate was 55.6% (95% confidence interval, 38.1–72.1%), and median progression-free survival was 7.7 months (95% confidence interval, 4.8–12.6 months). Grade 3 neutropenia was the most common haematological toxicity and occurred in 6.5% of 215 treatment courses. Grade 3 non-haematological toxicities included anorexia (1.4%) and diarrhoea (0.9%). There was no grade 4 toxicity of any kind.Conclusion:Our results suggest that this regimen is convenient, safe and promising, compared with conventional regimens for patients with metastatic colorectal cancer.

Overexpression of DRAM enhances p53-dependent apoptosis

Tumor suppressor p53‐dependent apoptosis is thought to be one of the most important tumor‐suppressive mechanisms in human tumorigenesis. Till date, “super p53” mutants exhibiting more potent ability to induce apoptosis than wild‐type p53 have been reported. These super p53s may provide a clue for development of novel therapeutic targets. However, the major mechanism underlying the super p53‐dependent apoptosis remains unclear. To identify critical gene(s) in this mechanism, we performed a comprehensive and comparative expression analysis in p53‐null Saos‐2 cells with conditional expression of wild‐type p53 and S121F, which was previously reported as a super p53 mutant. We identified damage‐regulated autophagy modulator (DRAM) as one of the genes that were more upregulated by S121F than wild‐type p53. Although knockdown of DRAM was not sufficient for reducing the ability of S121F to induce apoptosis, DRAM overexpression enhanced the ability in a wild‐type p53‐dependent manner. Here, we show that DRAM is an important gene for the enhancement of p53‐dependent apoptosis. Additional analysis of the mechanism of super p53‐dependent apoptosis may lead to the identification of novel drug targets for cancer therapy.

Phase II Trial of Cetuximab plus Irinotecan for Oxaliplatin- and Irinotecan-Based Chemotherapy-Refractory Patients with Advanced and/or Metastatic Colorectal Cancer: Evaluation of Efficacy and Safety Based on KRAS Mutation Status (T-CORE0801)

Background: Mutations in the KRAS gene have been identified as negative predictors of response to anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapy by patients with metastatic colorectal cancer (mCRC). However, it has been based on the study of mainly Caucasian mCRC patients. This prospective study investigated the relationship between the mutation status of EGFR-related genes including KRAS and the response rate (RR) to cetuximab plus irinotecan therapy in Japanese mCRC patients. Methods: Samples taken from 43 chemotherapy-refractory mCRC patients who had undergone cetuximab plus irinotecan therapy at 11 medical centers in Japan were subjected to direct DNA sequencing to determine the KRAS, BRAF, PIK3CA, NRAS, and AKT1 mutation status. The clinical outcome after the treatment was evaluated for each mutation status. Results:KRAS mutations were detected in 31.7% of 41 eligible patients. The RR to cetuximab plus irinotecan therapy was found to be 17.9 and 0% in the KRAS wild-type and mutant subgroups, respectively. Conclusion: Despite the identification of a lower-than-expected RR to treatment by the KRAS wild-type subgroup, KRAS mutation status appears to be a useful predictive marker of response to cetuximab plus irinotecan therapy in Japanese mCRC patients.

Upper Arm Central Venous Port Implantation: A 6-Year Single Institutional Retrospective Analysis and Pictorial Essay of Procedures for Insertion

Background The requirement of central venous (CV) port implantation is increasing with the increase in the number of cancer patients and advancement in chemotherapy. In our division, medical oncologists have implanted all CV ports to save time and consultation costs to other departments. Recently, upper arm implantation has become the first choice as a safe and comfortable method in our unit. Here we report our experience and discuss the procedure and its potential advantages. Methods All CV port implantations (n = 599) performed in our unit from January 2006 to December 2011 were analyzed. Procedural success and complication rates between subclavian and upper arm groups were compared. Results Both groups had similar patient characteristics. Upper arm CV port and subclavian implantations were equivalently successful and safe. Although we only retrospectively analyzed data from a single center, the upper arm group had a significantly lower overall postprocedural complication rate than the subclavian group. No pneumothorax risk, less risk of arterial puncture by ultrasound, feasibility of stopping potential arterial bleeding, and prevention of accidental arterial cannulation by targeting the characteristic solitary basilic vein were the identified advantages of upper arm CV port implantation. In addition to the aforementioned advantages, there is no risk of “pinch-off syndrome,” possibly less patient fear of manipulation, no scars on the neck and chest, easier accessibility, and compatibility with the “peripherally inserted central catheter” technique. Conclusions Upper arm implantation may benefit clinicians and patients with respect to safety and comfort. We also introduce our methods for upper arm CV port implantation with the videos.