A high-performance liquid chromatographic assay for CI-973, a new anticancer platinum diamine complex,in human plasma and urine ultrafiltrates

Summary CI-973 is a new platinum compound with antitumor properties that is currently undergoing phase II clinical trials. A high-performance liquid chromatographic (HPLC) assay was developed and validated for ultrafiltrates of human plasma and urine to support phase I clinical trials. Plasma ultrafiltrate (0.5 ml) was extracted using C18 solid-phase cartridges. Urine was diluted 10-fold and extracted first with SAX solid-phase cartridges and then with C18 cartridges. For both matrices, the eluate from the C18 cartridges was injected directly. A Whatman PAC 10 column (4.6×250 mm, 10-m particle size) and ultraviolet detection at 205 nm were used for both analyses. The mobile-phase buffer was 0.05m sodium perchlorate (pH 2.3). The mobile-phase acetonitrile: buffer ratio, column temperature, and flow rate were 8911 (v/v), 40°C, and 2.0 ml/min, respectively, for the plasma ultrafiltrate assay and 8515 (v/v), 50°C, and 1.0 ml/min, respectively, for the urine ultrafiltrate assay. Standard curves were linear from 0.25 to 500 g/ml and from 1.0 to 250 g/ml for the plasma and urine assays, respectively. The accuracy of the assay lay within 4.5% of the nominal values, and the precision was 6.2%; the recovery of CI-973 varied from 79.2% to 105%. CI-973 remains stable in plasma for at least 6 h, at room temperature, in ultrafiltrates of both matrices for at least 15 days at –72°C, and in water for at least 6 months at –72°C.     

Microsatellite instability and novel mismatch repair gene mutations in northern Chinese population with Hereditary non‐polyposis colorectal cancer

OBJECTIVE: Hereditary non‐polyposis colorectal cancer (HNPCC) syndrome is the most common cause of hereditary colorectal cancer with an early age of onset. Microsatellite instability (MSI) and germline mutation in one of the DNA mismatch repair (MMR) genes are found in the majority of HNPCC families and provide an opportunity for genetic diagnosis and prophylactic screening. The MMR gene mutation spectrum may vary across different populations and be influenced by founder mutations that prevail in specific ethnic groups. China is a big and ancient nation with enormous genetic diversity, which is especially notable between the northern and southern Chinese populations. A MMR gene mutation database for the southern Chinese population based in Hong Kong has been previously established. This study compares the MMR gene mutation spectrum and the MSI of HNPCC between the northern and southern Chinese populations. METHODS: Twenty‐five HNPCC families from northern China were systematically analyzed. The MSI analysis was performed using five loci in the USA National Cancer Institute (NCI) panel (D2S123, D5S346, BAT‐25, BAT‐26 and BAT‐40) by PCR from the tumor and normal tissue. MSH2, MSH6 and MLH1 were performed using immunohistochemical staining. Two founder mutations of MSH2 and MLH1 were examined by PCR base analyses using primers flanking the two deletion sites (c.1452_1455delAATG in MSH2 and 1.8 kb deletion involving exon 11 of MLH1) . RESULTS: Of the 25 families collected, 19 met Bethesda guideline (BG) 1 and six met BG3. Twenty‐two (15.7%) were extra‐colonic cancers with gastric cancer (in seven patients) being the most common cancer type. Of the 25 tumors analyzed, 21 (84%) were high level microsatellite instability (MSI‐H) and four (16%) were microsatellite stable (MSS). Eighteen (86%) of the 21 MSI‐H tumors showed loss of either the MLH1 or the MSH2 protein. Three MSI‐H tumors and all four MSS tumors showed no loss of expression of the three MMR proteins. Out of the 21 patients with MSI‐H tumors, 12 (57%) showed pathogenic germline mutations in either MLH1 (n = 8) or MSH2 (n = 4). Overall, three novel mutations (in patients H22, H17 and H29) have been identified. One of them, c.503_4insA, caused a frameshift mutation in the MLH1 gene. The other two were found in the MSH2 gene, including a frameshift (c.899_890insAT) and a splice junction (IVS7‐1G→A, SA of Exon 8) mutation. CONCLUSIONS: The results suggest a distinctly different mutation spectrum of MMR genes between northern and southern Chinese populations and call for a systematic, nationwide study to facilitate the design of a MMR gene mutation detection strategy tailored for individual populations in China.     

Coronarin D induces human oral cancer cell apoptosis though upregulate JNK1/2 signaling pathway

The incidence of oral cancer is increasing all over the world, with rates particularly high in Southeast Asian countries, such as Taiwan. Coronarin D (CD) has been confirmed to have anti‐inflammatory, anti‐bacterial effects, and anti‐apoptotic effects in human hepatocellular carcinoma and nasopharyngeal carcinoma. The purpose of this study is to explore whether CD has a suppression effect on oral cancer cells and the mechanisms involved. The results of our study revealed the significantly decreased cancer cell viability and increased activation of apoptosis via increased loss of mitochondrial membrane potential, increased death receptors, leading to the activation of caspase‐8, ‐9, ‐3. Moreover, the rate of apoptosis of cells treated with CD plus JNK inhibitors was decreased compared to CD‐treated cells. This is the first study to demonstrate that CD induces apoptosis in human oral cancer cells and can be expected to be a promising anticancer agent for oral cancer treatment.     

Atypical squamous cells of undetermined significance on cervical smears: follow-up study of an Asian screening population

BACKGROUND: The current study reports on the significance of cervical smears identified as atypical squamous cells of undetermined significance (ASCUS) in the largest Asian screening population to date. METHODS: From January 1998 to December 1999, 190,000 cervical smears were evaluated by the cervical cytology laboratory at the University of Hong Kong (Hong Kong, China). From these smears, 5579 ASCUS were identified. Follow-up cytology and histology findings were analyzed. RESULTS: Follow-up cytology or biopsy results were retrieved for 3601 women (64.5%). Of these, 544 (9.8%) and 96 women (1.7%) were found to have low-grade (LSIL) and high-grade (HSIL) squamous intraepithelial lesions, respectively. Biopsy results were obtained for 198 (36.4%) of the 544 women with LSIL. One hundred seventy-nine (32.9%) and 19 women (3.5%) were confirmed to have cervical intraepithelial neoplasia (CIN)-1 and CIN-2-CIN-3, respectively. Biopsy results were retrieved for 53 (55.2%) women with HSIL. Forty patients (41.7%) were confirmed to have CIN-2-CIN-3, whereas CIN-1 was found in the remaining patients. One woman with squamous cell carcinoma was diagnosed by colposcopic biopsy after immediate referral following a diagnosis of ASCUS. There was a significantly larger proportion of LSIL or HSIL (P < 0.0001) or higher-grade findings in women with ASCUS compared with the general screening population. Infective organisms were identified in 412 women (7.4%) with ASCUS. These women had a decreased risk of subsequent development of LSIL (P < 0.0001) or HSIL (P = 0.027). CONCLUSIONS: ASCUS smears indicated an increased risk of HSIL or carcinoma. The authors suggested careful patient follow-up in such cases.     

Aqueous Stability of Human Epidermal Growth Factor 1-48

Human epidermal growth factor 1-48 (hEGF 1-48, Des(49-53)hEGF) is a single chain polypeptide (48 amino acids; 3 disulfide bonds; 5445 Da) possessing a broad spectrum of biologic activity including the stimulation of cell proliferation and tissue growth. In this study, three primary aqueous degradation products of hEGF 1-48 were isolated using isocratic, reverse phase/ion-pair HPLC. The degradation products were characterized using amino acid sequencing, electrospray ionization mass spectrometry, isoelectric focusing, and degradation kinetics. Results indicate that hEGF 1-48 degrades via oxidation (Met²¹), deamidation (Asn¹), and succinimide formation (Asp¹¹). The relative contribution of each degradation pathway to the overall stability of hEGF 1-48 changes as a function of solution pH and storage condition. Succinimide formation at Asp¹¹ is favored at pH < 6 in which aspartic acid is present mostly in its protonated form. Deamidation of Asn¹ is favored at pH > 6. The relative contribution of Met²¹ oxidation is increased with decreasing temperature, storage as a frozen solution (–20°C), and exposure to fluorescent light.     

p53-R273H gains new function in induction of drug resistance through down-regulation of procaspase-3

Development of drug resistance is one of the major obstacles in cancer chemotherapy. The molecular mechanism leading to drug resistance is still not fully understood. A10A cells, a doxorubicin-resistant subline of human squamous cell carcinoma A431 cells, showed cross-resistance to methotrexate and also resistance to the drug-induced apoptosis. The cells also showed overexpression of a mutated form of p53, p53-R273H (Arg to His at codon 273), and down-regulation of procaspase-3. Knockdown of p53-R273H by p53 small interfering RNA in A431 cells increased procaspase-3 level and sensitized the cells to drug-induced apoptosis. On the other hand, transfection of p53-R273H into p53 null human osteosarcoma Saos-2 cells down-regulated procaspase-3 level and induced resistance to the drug toxicity and drug-induced apoptosis. The results support the idea that p53-R273H may gain new functions in induction of drug resistance and impairment in drug-induced apoptosis through down-regulation of procaspase-3 level. The study sheds new light on the understanding of the gain of function and drug resistance mechanisms associated with mutant p53.