Clinical significance of tumor mutation burden and DNA damage repair in advanced stage non-small cell lung cancer patients.

OBJECTIVE: This study aimed to investigate the impact of tumor mutational burden (TMB) and DNA damage repair (DDR) gene alteration on overall survival (OS) in advanced non-small cell lung cancer (NSCLC) patients. PATIENTS AND METHODS: A DNA library of cancer cells from 67 NSCLC patients in stages III-IV was constructed for next-generation sequencing (NGS). Geneseeq422 probes were used for hybridization enrichment. The target-enriched library was sequenced on HiSeqNGS platforms, and we analyzed the relevant signaling pathways. Then, we correlated the OS of the patients with TMB and DDR mutations. RESULTS: Many significant alterations were found, including in the EGFR, p53, KRAS, RB1, ERBB2, NF1, DNMT3A, ALK, MYC, PIK3CA, ROS1, BRAF, ARID1A, PTEN, CDKN2A, and FGF19 genes. We also identified many mutations in the genes relevant to the DDR pathway. Interestingly, we found that the TMB of patients with DDR gene mutations was dramatically higher than that in the DDR wild-type (WT). Univariable analysis showed that DNMT3A, RB1, DDR pathway-related gene mutations, and TMB were critical factors for the effects on OS. Multivariable analysis confirmed that DNMT3A and mutations in the DDR pathway-related genes were important for predicting OS. CONCLUSIONS: Multiple mutations in the genes of the DDR pathway caused higher TMB levels, which resulted in longer OS. By contrast, OS was significantly longer in patients with non-DNMT3A mutations than in those with DNMT3A variants. DNMT3A alteration in NSCLC patients led to poor outcomes.

In vitro inhibition of invasion and metastasis in colon cancer cells by TanIIA.

The purpose of this study was to investigate the effect of the traditional Chinese medicine TanIIA on the viability, invasion, and metastasis of SW480 cells. SW480 cells were treated with TanIIA for 24 h, and MTT assays were performed to determine the effect of TanIIA on cell viability. Transwell transmembrane experiments were applied to test the effect of 1.0 mg/mL TanIIA on SW480 cell invasion and metastasis abilities. Western blotting was performed to determine the expression of the tumor cell metastasis proteins E-cadherin, vimentin, and MMP-9. The cell growth inhibition rates were 0%, 26 ± 4.3%, 43.47 ± 4.0%, 63.0 ± 5.5%, and 76.8 ± 7.8% for treatment with 0, 0.5, 1.0, 2.0, and 5.0 mg/L TanIIA, respectively. The differences in the cell viability inhibitory rates among all groups were statistically significant (P < 0.05). The Transwell assay results indicated that SW620 cell invasion and metastasis abilities were strongly inhibited by 1.0 mg/mL TanII. The western blotting results showed that the expression of E-cadherin was significantly increased and that the expression levels of vimentin and MMP-9 were significantly decreased after treatment with 1.0 mg/mL TanII for 24 h (P < 0.05). Tan II can effectively inhibit the biological activity of colon cancer in vitro and prevent the invasion of colon cancer cells.

Effect of preconditioning with hyperbaric oxygen on neural cell apoptosis after spinal cord injury in rats.

AIM: The aim of this study was to study the effect of preconditioning with hyperbaric oxygen on neural cell apoptosis after spinal cord injury in rats at different times, and to study the mechanism of neuroprotection with hyperbaric oxygen preconditioning after spinal cord injury. METHODS: Fifty-five adult Sprague-Dawley rats were randomly divided into 3 groups, a hyperbaric oxygen preconditioning group (Hoping, N.=25), a normal injury group (NI, N.=25) and a control group (CON, N.=5). The acute spinal cord injury rat models were established using Allen's method, the spinal cord injury selections were obtained separately after injury day 1, 5, 7, 10 and 14, the neural cell apoptosis after the spinal cord injury in the rat was detected by the HE staining and TUNEL method. RESULTS: The TUNEL-positive apoptotic cells were found in both the hyperbaric oxygen preconditional group and in the normal injury group. However, There was a statistically significant difference between each group (P<0.05). In the hyperbaric oxygen preconditioning group, the number of apoptotic cells decreased, while the neurofunction of the spinal cord was improved compared with the other two groups. CONCLUSION: HBO preconditioning can reduce the number of apoptotic cells and promote the nerve functional recovery in rats after spinal cord injury, which provide some experimental basis for currently clinical hyperbaric oxygen therapy.

Cucurbitacin IIa: a novel class of anti-cancer drug inducing non-reversible actin aggregation and inhibiting survivin independent of JAK2/STAT3 phosphorylation.

BACKGROUND: Cucurbitacin (Cuc) and triterpene-derived natural products exhibit anti-cancer potential in addition to their conspicuous anti-bacterial and anti-inflammatory activity. Recently, inhibition of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling was shown to underlie the effects of Cuc family on inducing cell death in cancer. METHOD: We purified Cuc IIa, the active component from the medicinal plant Hemsleya amalils Diels, which shows different structural modifications from other Cuc derivatives. We investigated the mechanisms of its inhibitory effects on cancer cells in vitro and tumour growth in vivo. RESULTS: Cuc IIa induced the irreversible clustering of filamentous actin and arrested cell cycle by the increases in G2/M populations. Cuc IIa resulted in the reduced phospho-Histone H3 and markedly increased cleavage of poly-(ADP-ribose) polymerase or PARP, immediate upstream of DNA breakdown as the result of caspase activation, consistent with mitotic blockage-induced cell death. However, unlike other Cuc members, Cuc IIa did not suppress JAK2/STAT3 phosphorylation or alter phosphorylation of mitogen-activated protein kinases. Instead, the expression of the cell cycle-regulated Inhibitor of Apoptosis Protein (IAP) survivin was reduced. Introducing oncoprotein δ-catenin, which increased survivin expression and suppressed small GTPase RhoA, reduced efficacy of Cuc IIa to induce cell death. Supporting the effects of Cuc IIa on actin cytoskeletal signaling, RhoA phosphorylation was reduced suggesting its increased activity. CONCLUSION: Cuc IIa is a novel class of anti-cancer drug in suppression of cancer cell expansion by disrupting the actin cytoskeleton and directing the cell to undergo PARP-mediated apoptosis through the inhibition of survivin downstream of JAK2/STAT3.

Telomerase inhibition in cancer therapeutics: molecular-based approaches.

Current standard cancer therapies (chemotherapy and radiation) often cause serious adverse off-target effects. Drug design strategies are therefore being developed that will more precisely target cancer cells for destruction while leaving surrounding normal cells relatively unaffected. Telomerase, widely expressed in most human cancers but almost undetectable in normal somatic cells, provides an exciting drug target. This review focuses on recent pharmacogenomic approaches to telomerase inhibition. Antisense oligonucleotides, RNA interference, ribozymes, mutant expression, and the exploitation of differential telomerase expression as a strategy for targeted oncolysis are discussed here in the context of cancer therapeutics. Reports of synergism between telomerase inhibitors and traditional cancer therapeutic agents are also analyzed.

Cytotoxicity and NMR spectral assignments of ergolide and bigelovin.

Two potent cytotoxic sesquiterpene lactones, ergolide (1) and bigelovin (2) were isolated from Inula hupehensis I. helianthus-aquatica and their structures and NMR data were assignment unambiguously by using a combination of one-and two-dimensional NMR techniques and computer modeling calculations.

Expression of selected osteogenic markers in the fibroblast-like cells of rat marrow stroma.

The fibroblast-like cells in the marrow stromal system were separated from endothelial cells and macrophages by negative selection of magnetic beads. Immunocytochemistry confirmed that these fibroblast-like cells expressed fibronectin and collagen Type III, but not Factor VIII and epithelial membrane antigen (endothelial cell markers) or Mac I (macrophage marker). The fibroblast-like stromal cells (FSC) synthesized the insulin-like growth factors (IGF)-I and -II in amounts equivalent to that produced by unfractionated marrow stromal cells (UMSC); in both, the concentration of IGF-II was 10 times higher than that of IGF-I. Northern analysis revealed that FSC and UMSC expressed identical patterns of mRNAs for IGF-I and transforming growth factor (TGF) -beta 2, for osteopontin, and for procollagen Types I and III (Type I > Type III). Type II procollagen mRNA was not expressed in both cell populations. The TGF-beta 2 gene mRNA was expressed at a lower level by the FSC than UMSC. The pattern of gene expression in these cells is consistent with an osteoprogenitor phenotype. Both FSCs and UMSCs express parathyroid hormone (PTH) and estrogen receptor genes (rtPCR technique). The study provides additional evidence that fibroblast-like marrow stromal cells have an osteoblast signature, and that they are largely responsible for the osteogenic performance of cells in unfractionated marrow.

Effects of C-terminal parathyroid hormone-related peptide on osteoblasts.

A C-terminal analog of parathyroid hormone-related peptide (PTHrP), PTHrP(107-139), was found to stimulate cAMP production in three osteoblast cell preparations. The effect was studied most extensively in ROS 17/2.8 cells. The effect was dose-related and comparable in magnitude to that produced by PTHrP(1-34), but potency was lower. The functional significance of the cAMP effect is unknown, but preliminary findings indicated that PTHrP(107-139) also inhibited osteopontin mRNA levels in ROS 17/2.8 cells treated with peptide for 48 h. The results suggest that the carboxy-terminal region of PTHrP may play a role in bone metabolism by influencing osteoblast activity.

Differential effects of new breviscapine on arachidonic acid metabolisms in blood cells and endothelial cells.

New breviscapine (NB) is the soluble sodium and calcium salts of 4'-scutellarin-7-glucuronide extracted from the Chinese herb Erigeron breviscapus (Vant) Hand-Mazz. It inhibited platelet thromboxane B2 production without alteration of HETE. It also inhibited 6-ketoprostaglandin F1 alpha production by endothelial cells. For leukocytes, NB did not affect thromboxane B2 production. However, it potentiated the effect of calcimycin in stimulating LTB4 formation. These results indicate that NB exerts differential effects on the arachidonic acid metabolism in different blood cells and endothelial cells.

Pharmacokinetics and tissue distribution of 2-fluoro-beta-alanine in rats. Potential relevance to toxicity pattern of 5-fluorouracil.

Clinical pharmacokinetic studies in our laboratory demonstrated that 2-fluoro-beta-alanine (FBAL), the major catabolite of fluorouracil (FUra), has a prolonged elimination with an approximately 150-fold longer half-life than that of the unchanged drug in humans [Heggie et al.: Cancer Res. 47, 2203-2206 (1987)]. Recent studies have suggested that FUra catabolites, such as FBAL, may have a role in neurotoxicity and cardiotoxicity that may occur during FUra chemotherapy [Okada et al.: Acta Neuropathol. 81, 66-73 (1990)]. This study was undertaken to determine the kinetics and tissue distribution of FBAL in rats following iv bolus administration of radiolabeled FBAL. Plasma disappearance curves for FBAL could be described by the sum of three exponentials, with half-lives of 0.26, 12.1, and 8426 min. Radioactivity, consisting mainly of FBAL-bile acid conjugates, was excreted in bile within 30 sec of iv bolus administration of FBAL and continued throughout the experimental period at concentrations 10-100-fold higher than that of the corresponding plasma level. Urinary excretion, consisting mainly of free FBAL, represented the major pathway of elimination of FBAL, with 40% of the administered dose excreted within 24 hr and approximately 70% over 192 hr. Fecal excretion was a minor pathway of elimination of FBAL, with approximately 10% of the administered dose excreted over 192 hr. During the initial 30 min, the highest levels of tissue radioactivity were found in the kidneys, liver, spleen, lungs, and heart. Radioactivity was retained over longer time periods in the enterohepatic circulation, central nervous system, heart, and skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of marrow stromal cells to the regulation of osteoblast proliferation in rats: evidence for the involvement of insulin-like growth factors.

Fibroblast-like marrow stromal cells are believed to play a role in the maintenance of osteoblast populations at the marrow-bone interface. We now report that this interaction may be very specific. Stromal cell conditioned medium (SC-CM) stimulated DNA synthesis and proliferation in culture of neonatal rat calvarial osteoblasts at low concentrations (1.25-5%), but was inhibitory at 10%. At growth promoting effective concentrations, the activity of osteoblast alkaline phosphatase was decreased. This action was selective since SC-CM failed to promote the growth of rat calvarial fibroblasts. Characterization of the SC-CM indicated the cells produced IGF-I and -II and a wide range of molecular weight fractions with putative stimulatory action (FPLC analysis using Superose 12 and 6 gel permeation columns). HPAE-PAD analysis showed that some elements were glycosylated, and the composition suggested the presence of N- and O-linked oligosaccharide chains. Because rat marrow stromal fibroblast-like cells produce a number of osteotropic factors which affect calvarial osteoblast growth, these interactions may be important to considerations about the etiology of the osteoporoses.

Inhibition of fluoropyrimidine catabolism by benzyloxybenzyluracil. Possible relevance to regional chemotherapy.

Regional infusion with fluoropyrimidines is useful in the treatment of hepatic metastases. However, the effectiveness of regional infusion is minimized by rapid degradation of 5-fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd) by the liver which limits the availability of drug for anabolism to active metabolites. 5-Benzyloxybenzyluracil (BBU) is a potent inhibitor of dihydropyrimidine dehydrogenase (DPD), the initial enzyme in FUra catabolism (Naguib FMN, el Kouni MH and Cha S, Biochem Pharmacol 38: 1471-1480, 1989). The effect of BBU on fluoropyrimidine catabolism in the liver was evaluated using the isolated perfused rat liver (IPRL). BBU infused at 0.35 microM over the course of 1 hr demonstrated no hepatotoxicity as measured by bile flow, O2 uptake and lactate dehydrogenase leakage. The effect of BBU (0.35 microM) on the catabolism of FUra (10 microM) or FdUrd (10 microM) was quantitated by HPLC at 5- or 10-min intervals over a 1-hr period. BBU maximally inhibited FUra catabolism by approximately 83%. Further studies utilizing short-term (20 min) infusion of BBU prior to administration of FUra suggested that the inhibition of DPD was reversible. While FdUrd phosphorolysis was not affected, subsequent catabolism of FUra decreased by 70%. Studies on isolated hepatocytes indicated that the increased FUra level in perfusate resulted from inhibition of FUra catabolism and not from inhibition of FUra transport. The significant inhibition of FUra catabolism suggests that BBU may be useful in modulating regional chemotherapy by these fluoropyrimidines.

[Drug evaluation of huperzine A in the treatment of senile memory disorders].

Huperzine A is an alkaloid which was first isolated from Huperzia serrata (Thumb) Trev by Zhejiang Academy of Medical Sciences and Shanghai Institute of Materia Medica, Chinese Academy of Sciences. It exhibits a significant anticholinesterase activity and has been used on myasthenia gravis patients. The therapeutic effects were studied by random, match and double-blind method on 56 patients of multi-infarct dementia or senile dementia and 104 patients of senile and presenile simple memory disorders. The curative effects were evaluated by Wechsler memory scale. The im dose for multi-infarct dementia was 0.05 mg bid for 4 wk, whereas that for senile and presenile simple memory disorders was 0.03 mg bid for 2 wk. Saline was used on control group. The result showed that the curative effect of huperzine A was significant. Only a few patients felt slight dizziness and this did not affect the therapeutic effects.

Partial characterization of rat marrow stromal cells.

Fibroblast-like rat marrow stromal cell (CFU-F) cultures have been characterized in terms of their responsiveness to calciotropic hormones, metal ions, the nonsteroidal antiinflammatory drug, and by their putative paracrine role in the maintenance of active populations of osteoblasts at the marrow-bone interface. These studies indicate that CFU-Fs lack a complete osteoblast signature. Subconfluent CFU-Fs grown in the presence or absence of 10(-7) M dexamethasone lack receptors for PTH and calcitonin, and fail to show enhanced cAMP or cGMP responses to 10(-7) M 1-34 PTH (rat), or any evidence of osteocalcin production [+/- 10(-9) M 1,25-(OH)2D3]. Low concentrations of fluoride [10(-12) and 10(-9) M] stimulated CFU-F grown in vitro in serum-free media, though higher levels (10(-7) and 10(-6) M), inhibited growth in vivo and in vitro. Aluminum (10(-12)-10(-7) M) and ibuprofen (10(-7) M) did not alter normal growth patterns, indicating an action on bone cells more differentiated than CFU-Fs. Serum-free conditioned medium (CM) from control and ovariectomized (OVX)/OVX+ dihydrotachysterol-Rx rat CFU-F cultures was mitogenic for neonatal rat calvarial osteoblasts in vitro, but not for ROS 17/2.8 cells. The studies affirm the mesenchymal-like character of CFU-Fs and project their significant role in sustaining functional endosteal osteogenic cell populations.

Disposition and metabolism of 2-fluoro-beta-alanine conjugates of bile acids following secretion into bile.

Since 2-fluoro-beta-alanine (FBAL) conjugates of bile acids (BA), the primary biliary metabolites of fluoropyrimidine (FP) drugs, have been suggested to be related to the hepatotoxicity which develops in patients receiving FP chemotherapy by intrahepatic arterial infusion (Proc. Natl. Acad. Sci. USA 84, 5439-5443, 1987), it was important to determine whether they undergo enterohepatic circulation and hence accumulate in the liver and biliary system. In initial studies, sensitivity of FBAL-BA conjugates to hydrolysis by pancreatic enzymes was examined. In subsequent in vivo studies, a model FBAL-BA conjugate, FBAL-chenodeoxycholate (FBAL-CDC), was introduced into the lumen of the small intestine of anesthetized rats with biliary fistulas to quantitate the intestinal absorption, metabolism and tissue distribution of the conjugate. The results indicated that: (1) FBAL-BA conjugates were resistant to hydrolysis by pancreatic enzymes (carboxypeptidase A, carboxypeptidase B and trypsin) and by human pancreatic juice, but were completely hydrolyzed by cholyglycine hydrolase. (2) At least one-half of the administered FBAL-CDC was deconjugated during the process of intestinal absorption, as shown by HPLC analysis of the radioactivity in portal venous blood. (3) Deconjugated FBAL or CDC was reconjugated in liver with other bile acids or amino acids (glycine and taurine), respectively, as shown by radiochromatography of bile. (4) FBAL, formed as a result of hydrolysis of FBAL-CDC, had a wide tissue distribution. In conclusion, FBAL-CDC has a rapid turnover during its enterohepatic circulation due to deconjugation in the intestine and reconjugation in the liver.

Microorganisms capable of metabolizing the herbicide metolachlor.

We screened several strains of microorganisms and microbial populations for their ability to mineralize or transform the herbicide metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)-acetami de] because such cultures would potentially be useful in the cleanup of contaminated sites. Although we used various inocula and enrichment culture techniques, we were not able to isolate microorganisms that could mineralize metolachlor. However, strains of Bacillus circulans, Bacillus megaterium, Fusarium sp., Mucor racemosus, and an actinomycete were found to transform metolachlor. Several metabolites could be determined with high-performance liquid chromatography. The tolerance of the strains to high concentrations of metolachlor was also evaluated for the usefulness of the strains for decontamination. Tolerance of the actinomycete to metolachlor concentrations over 200 ppm (200 micrograms/ml) was low and could not be increased by doubling the sucrose concentration in the growth medium or by using a large biomass as inoculum. However, a Fusarium sp. could grow and transform metolachlor up to a concentration of 300 ppm.

Spermicidal effect in vitro by the active principle of garlic.

The in vitro spermicidal effect of Allitridum, an active principle of garlic, was investigated. The data showed that sperm motility was inhibited with various concentrations of Allitridum at different intervals ranging from 20 seconds-200 minutes as compared to control. An obvious immobilization of spermatozoa occurred at 7.5 mg/ml of Allitridum. The effects on sperm motility appeared to be dose-dependent.

PIP: The in vitro spermicidal effect of Allitridum, an active principle of garlic, was investigated. The data showed that sperm motility was inhibited with various concentrations of Allitridum at different intervals ranging from 20 seconds-200 minutes as compared to control. An obvious immobilization of spermatozoa occurred at 7.5 mg/ml of Allitridum. The effects on sperm motility appeared to be dose-dependent. Male rats and hamsters were used for the study as well as human spermatozoa.