Oridonin induces apoptosis of HeLa cells via altering expression of Bcl-2 ／ Bax and activating caspase-3 ／ ICAD pathway

INTRODUCTIONHerbal medicine, Donglingcao (rabdosiarubescens), has been traditionally used in China for thetreatment of various diseases such as leukemia.Oridonin (Fig 1) is a diterpenoid compound isolated fromRabdosia rubescens (hemsl). It has various pharmaco-logical and physiological effects such as anti-inflammation, anti-bacteria, anti-tumor[1-3] and has beenused for the treatment of human cancers, especiallyFig 1. Chemical structure of oridonin.esophageal carcinoma[4]. This comp…     

Atypical apoptosis in L929 cells induced by evodiamine isolated from Evodia rutaecarpa

Two alkaloids, evodiamine and rutaecarpine, isolated from the dried fruits of Evodia rutaecarpa Bentham were evaluated in vitro for antiproliferation activity on tumor cells versus human peripheral blood mononuclear cells (PBMC). Evodiamine had more potent cytotoxic effects on five tumor cell lines (human malignant melanoma A375-S2, human cervical cancer HeLa, human breast adenocarcinoma MCF7, human acute monocytic leukemia THP-1, murine fibrosarcoma L929) than rutaecarpine. Moreover, evodiamine did not affect PBMC viability for a 36 h culture period. Although apoptotic bodies were observed in evodiamine-treated L929 cells stained with Hoechst 33258, DNA fragmentation as a hallmark of apoptosis was not found. Caspases were involved in the protection of L929 cells against cell death. Evodiamine initiated atypical apoptosis in L929 cells by cycle arrest at the G0/G1 phase.     

Quantitative and qualitative comparison of virulence traits,including murine lethality,among different M types of group A streptococci

Epidemiological studies have proposed an association between group A streptococci (GAS) bearing a particular M serological type and pathologic conditions such as streptococcal toxic shock syndrome (STSS). M1 and M3 GAS are isolated from STSS cases more frequently, whereas M4 and M12 GAS are isolated from non-STSS cases more frequently. To investigate whether there is any difference contributing the M-type association among GAS, we compared various virulence traits, including the murine lethality of M4, M12, M1, and M3 GAS clinical isolates, which are not clonally related to one another. Murine lethality, the activities of superantigens, streptolysin O, and nicotinamide adenine dinucleotide glycohydrolase, and the presence of the speA and speC genes were closely associated with M type. These results indicate that M types may serve, in part, as markers for strains/clones with particular profiles of virulence traits and mouse lethality.     

Usefulness of arbitrarily primed polymerase chain reaction (AP-PCR) method for DNA fingerprinting analysis of Mycobacterium tuberculosis (MTB); comparison between DNA restriction fragment-length polymorphism (RFLP) method and AP-PCR method

We have been analyzing cases suspected as outbreak of Mycobacterium tuberculosis in Tokyo using RFLP method. This time we analyzed 27 strains of MTB from 5 cases in two hospitals, a family, member of social activity and stuff of a corporation using both RFLP and AP-PCR methods. At 4 cases, over 80% of strains were same pattern in each cases with RFLP and AP-PCR and were identified as a patient to patients transmission of MTB. At one case, in a hospital, each strains were completely different patterns at both methods, which showed it was not a outbreak case. Results of RFLP and AP-PCR were completely same, which indicates AP-PCR is also useful and rapid method for epidemiological analysis of MTB infection as well as RFLP.     

Frameshift mutations and a length polymorphism in the hMSH3 gene and the spectrum of microsatellite instability in sporadic colon cancer.

Mutations in the hMVSH3 gene in sporadic colon cancer with microsatellite instability (MSI) were investigated, since several mismatch repair genes were known to be mutated in cancers with MSI, but only deletions in the (A)8 region in the hMSH3 gene have been reported. We also analyzed the relationships between hMSH3 mutations and the spectrum of MSI. We screened MSI in 79 sporadic colon cancer samples using mono- and dinucleotide repeat markers and the samples with MSI were further analyzed for tri- and tetranucleotide repeat instability and mutations in the hMSH3 gene by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Five (6%) out of 79 tumors were MSI-H and 15 (19%) were MSI-L. Two MSI-H tumors showed insertion in the (C)8 region in the hMSH6 gene and one tumor showed insertion and deletion in the (A)8 region in the hMSH3 gene, and two of the three above tumors showed MSI in tri-and tetranucleotide repeats. One MSI-L tumor showed somatic alteration in a 9-bp repeat sequence in hMSH3. No frameshift mutations were found in the (A)7 and (A)6 regions in hMSH3. Thus, we confirmed that the (A)8 region in hMSH3 is a hot spot and mutations in the (A)7 and (A)6 regions in hMSH3 are not common. The hMSH3 mutation may enhance genomic instability in some colorectal cancers.     

Frameshift Mutations and a Length Polymorphism in the hMSH3 Gene and the Spectrum of Microsatellite Instability in Sporadic Colon Cancer

Mutations in the hMSH3 gene in sporadic colon cancer with microsatellite instability (MSI) were investigated, since several mismatch repair genes were known to be mutated in cancers with MSI, but only deletions in the (A)₈ region in the hMSH3 gene have been reported. We also analyzed the relationships between hMSH3 mutations and the spectrum of MSI. We screened MSI in 79 sporadic colon cancer samples using mono- and dinucleotide repeat markers and the samples with MSI were further analyzed for tri- and tetranucleotide repeat instability and mutations in the hMSH3 gene by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Five (6%) out of 79 tumors were MSI-H and 15 (19%) were MSI-L. Two MSI-H tumors showed insertion in the (C)₈ region in the hMSH6 gene and one tumor showed insertion and deletion in the (A)₈ region in the hMSH3 gene, and two of the three above tumors showed MSI in tri-and tetranucleotide repeats. One MSI-L tumor showed somatic alteration in a 9-bp repeat sequence in hMSH3. No frameshift mutations were found in the (A)₇ and (A)₆ regions in hMSH3. Thus, we confirmed that the (A)₈ region in hMSH3 is a hot spot and mutations in the (A)₇ and (A)₆ regions in hMSH3 are not common. The hMSH3 mutation may enhance genomic instability in some colorectal cancers.     

Prolonged lung retention of 123I-IMP in pulmonary disease

The accumulation of venously injected ¹²³I-IMP in the lung was studied. Between 30 and 50 min after the injection of the 1.5 mCi ¹²³I-IMP, the concentration of ¹²³I-IMP in the broncho-alveolar lavage fluid were much higher than in the blood. It was considered that ¹²³I-IMP was transported into the alveolar spaces and was absorbed by the alveolar cells. The half time (T _1/2) of the ¹²³I-IMP release from the lung between 10 and 25 min immediately after the injection was calculated. In normal subjects the T _1/2 ranged between 25 and 44 min and was prolonged in subjects with pulmonary fibrosis, sarcoidosis, and allergic alveolitis. It was considered that the retention of ¹²³I-IMP was related not only to the endothelial cells, but also to the alveolar cells. It was considered that the analysis of the lung release of ¹²³I-IMP forms a new lung dysfunction index.     

Silibinin activated p53 and induced autophagic death in human fibrosarcoma HT1080 cells via reactive oxygen species-p38 and c-Jun N-terminal kinase pathways

Our previous research demonstrated that hepatic-protectant silibinin induced autophagy in human fibro-sarcoma HT1080 cells through reactive oxygen species (ROS) pathway. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced autophagy and reversed silibinin's growth-inhibitory effect; besides, PFT-α decreased the activation of caspase-3, a crucial executor of apoptosis. Silibinin upregulated expression of p53/phosphorylated-p53 (p-p53) in a time-dependent manner. Catalase (scavenger of H(2)O(2)), superoxide dismutase (SOD) (scavenger of O(2)(?-)), and SB203580 (inhibitor of p38) attenuated upregulation of p53 expression, suggesting that p53 might be partially regulated by ROS-p38 pathway. On the other hand, c-Jun N-terminal kinase (JNK) increased autophagic death in silibinin-treated cells, and JNK/p-JNK expression was upregulated by silibinin time-dependently. Inhibition of JNK by SP600125 did not influence generation of ROS. Scavengers of H(2)O(2) or O(2)(?-) showed no effect on expression of JNK/p-JNK, indicating that JNK might not correlate with ROS in this process. However, activation of p53 was suppressed by SP600125; therefore the function of p53 was possibly controlled by JNK as well. Western blotting analysis showed that PFT-α reduced activation of extracellular regulated kinase1/2 (ERK1/2) and expression of protein kinase B (PKB, or Akt)/p-Akt. PD98059 (inhibitor of mitogen-activated protein kinase kinase (MEK)/ERK) and wortmannin (inhibitor of phosphoinositide 3-kinase (PI3K)/Akt) enhanced silibinin's cytotoxicity. Wortmannin augmented silibinin-induced autophagy, while PD98059 did not affect autophagic ratio. These results suggest that silibinin might induce p53-mediated autophagic cell death by activating ROS-p38 and JNK pathways, as well as inhibiting MEK/ERK and PI3K/Akt pathways.