Benzyl isothiocyanate induces protective autophagy in human lung cancer cells through an endoplasmic reticulum stress-mediated mechanism.

Isothiocyanates, such as allyl isothiocya¬nate (AITC), benzyl isothiocyanate (BITC), phenethyl isothio¬cyanate (PEITC) and sulforaphane (SFN), are natural compounds abundant in cruciferous vegetables, which have substantial chemopreventive activities against various human malignancies. However, the mechanisms underlying the inhibition of tumor cell growth by isothiocyanates are not fully understood. Since autophagy has dual functions in cancer, in the present study we investigated the effects of BITC on autophagy induction in human lung cancer cells in vitro and in vivo. BITC (1-100 µmol/L) dose-dependently inhibited the growth of 3 different human lung cancer cell lines A549 (adenocarcinoma), H661 (large cell carcinoma) and SK-MES-1 (squamous cell carcinoma) with IC50 values of 30.7±0.14, 15.9±0.22 and 23.4±0.11 µmol/L, respectively. BITC (10-40 µmol/L) induced autophagy in the lung cancer cells, evidenced by the formation of acidic vesicular organelles (AVOs), the accumulation of LC3-II, the punctate pattern of LC3, and the expression of Atg5. Pretreatment with the autophagy inhibitor 3-MA (5 mmol/L) significantly enhanced the BITC-caused growth inhibition in the lung cancer cells. Furthermore, BITC (20-40 µmol/L) activated ER stress, as shown by the increased cytosolic Ca2+ level and the phosphorylation of the ER stress marker proteins PERK and eIF2α in the lung cancer cells. Pretreatment with the ER stress inhibitor 4-PBA (5 mmol/L) attenuated the autophagy induction and potentiated the BITC-induced cell growth inhibition. In nude mice bearing A549 xenografts, administration of BITC (100 mg·kg-1·d-1, ip) for 8 weeks markedly suppressed the lung tumor growth, and significantly enhanced both autophagy and ER stress in the tumor tissues. Our results demonstrate that BITC inhibits human lung cancer cell growth in vitro and in vivo. In addition, BITC induces autophagy in the lung cancer cells, which protects the cancer cells against the inhibitory action of BITC; the autophagy induction is mediated by the ER stress response.

[Effect of m-nisoldipine on the Ca2+/CaM/CaN signal pathway in 5-HT-induced proliferation of rat PASMCs].

This study is to explore the activation of the Ca2+/CaM/CaN signal pathway in 5-HT-induced proliferation of rat pulmonary artery smooth muscle cells (PASMCs) and the inhibitory effect of m-nisoldipine (m-Nis) on this pathway. PASMCs were cultured with the explant technique. The proliferation of PASMCs was evaluated by MTT assay. Confocal microscopy was used to measure the change of [Ca2+]i. The mRNA expression of CaM and CaN was evaluated by RT-PCR and the activity of CaN was measured according to the instruction of kits. The results of MTT assay suggested that 5-HT (1 micromol x L(-1)) significantly induced the proliferation of rat PASMCs (P < 0.01), which was inhibited obviously by m-Nis (P < 0.05 or P < 0.01). Similarly, m-Nis inhibited 5-HT-induced elevation of [Ca2+]i (P < 0.01). The mRNA expression of CaM, CaN and the activation of CaN were also inhibited by m-Nis at different degrees (P < 0.05 or P < 0.01). Thus, the results of this study suggested that Ca2+/CaM/CaN signal pathway played an important role in 5-HT-induced proliferation of rat PASMCs, the inhibition of m-Nis on proliferation of rat PASMCs may be related to the blockage of Ca2+/CaM/CaN signal pathway by inhibiting the elevation of [Ca2+]i.

[Studies on preparation and dissolution test in vitro of sustained-release dropping pills of curcumin].

OBJECTIVE: To study the prescription and technique of sustained-release dropping pills of curcumin and inspect their release property in vitro. METHODS: The orthogonal test was used to screen the prescription and technique which were definited with the colligation evaluation of release and formation of dropping pills. RESULTS: The optimization of prescription and technique were as follows: stearic acid 70 mg, glycery monostearate 25 mg, solutol 6 mg, viscosity of cooling liquid was 100 mm2/s; the temperature of material liquid was 80 degrees C; the cooling temperature was 30 - 0 degrees C; the dropping speed was (21 +/- 2) dripping/min. The release behavior of sustained-release dropping pills of curcumin coincidented with Higuchi equation well and the character of sustained-release was transparent. CONCLUSIONS: The sustained-release dropping pills of curcumin have good property of sustained-release in vitro and their release behavior in vivo need to be inspected.

[The synergistic and toxicity-reducing effects of zhenqifuzheng injection on mice with 60Co radiotherapy].

OBJECTIVE: To study the synergistic and toxicity-reducing effect of zhenqifuzheng (ZQFZ) injection on mice with 60Co radiotherapy. METHODS: The tumor-bearing mice models with Hepatoma-22 were established. Mice in 60Co control group were treated by 60Co; ZQFZ injection were administered with dosages of 1.5, 3.0, 6.0 g/(kg x d) through ip for 7 days,then the inhibitory rate, the number of WBC,the spleen and thymus index were calculated. Meanwhile,the MDA content and SOD activity in serum were determined. Phagocytosis of mononuclear macrophage was determined with carbon particle clearance test. RESULTS: Each dose of ZQFZ injection inhibited the development of tumor, and had synergism with 60Co. High dose of ZQFZ injection increased the number of WBC, and the spleen index in all ZQFZ groups increased (P < 0.05 or P < 0.01). Whereas, no significant difference was found of thymus index in all groups, and the changes of MDA content and SOD activity induced by 60Co were reversed obviously by three doses of ZQFZ (P < 0.05 or P < 0.01). Meanwhile, non-specific immunity was enhanced in different degree in three doses of ZQFZ groups (P < 0.01). CONCLUSION: ZQFZ injection can increase the activity of non-specific immunity, improve the anti-tumor effects and meanwhile attenuate the toxicity of 60Co, which may be related to the antagonistic effect on lipid peroxidation.

Mechanisms for steep pulse irreversible electroporation technology to kill human large cell lung cancer cells L9981.

To explore the mechanisms for steep pulse irreversible electroporation technology to kill the lung cancer cell L9981. The apoptosis, cells mitochondrial membrane potential, internal PH changes and the intra-cellular calcium ions concentration were detected after steep pulses acted on the human large cell lung cancer cell L9981. Apoptosis test results indicated that cancer cells mainly experienced necrosis and apoptosis. Along with the increase of electric parameters, the proportion of the necrotic cells increased rapidly; the detection of cells mitochondrial membrane potential indicated that membrane potential occurred depolarization. Steep pulse can cause cancer cells to produce death and apoptosis .The PH value indicated that intracellular PH level down jumped. Internal PH became more acidic and led to cell death. The detection of intra-cellular calcium ions concentration showed that the number of free calcium significantly increased, and this change had killing effects on cell death and apoptosis. Steep pulse could induce cell apoptosis.

Apoptosis induced by benzyl isothiocyanate in gefitinib-resistant lung cancer cells is associated with Akt/MAPK pathways and generation of reactive oxygen species.

Gefitinib is the first targeted drug approved for non-small cell lung cancer (NSCLC) treatment. Clinical trails showed that patients with certain clinical and histologic characteristics (such as women, patients of East Asian descent, no history of smoking, and adenocarcinoma) had higher rates of response and overall survival. Despite excellent clinical response to gefitinib in certain NSCLC patients, nearly all patients who respond initially to gefitinib later develop drug resistance. Isothiocyanates have been shown to possess antitumor activity, inhibiting several types of cancer cells growth. However, there are limited studies on their effects on chemoresistance of cancer cells. In this report, we found that benzyl isothiocyanate (BITC) inhibited gefitinib-resistant human NSCLC cells growth by inducing apoptosis in a dose-dependent manner, and activated caspase-3. There were no effects of BITC on epidermal growth factor receptor and multidrug resistant proteins expression. BITC caused cell cycle arrest at G2/M phase, reactive oxygen species generation, and glutathione depletion. Akt activity and NFκB transcriptional activation were suppressed; mitogen-activated protein kinase and activator protein 1 (AP-1) were activated. Our results demonstrated that BITC overcame gefitinib resistance in lung cancer cells. The further understanding of the anti-resistance mechanism of BITC would contribute to establish it as a potent lead compound for the synthesis of novel anticancer drugs.

A polymorphic 3'UTR element in ATP1B1 regulates alternative polyadenylation and is associated with blood pressure.

Although variants in many genes have previously been shown to be associated with blood pressure (BP) levels, the molecular mechanism underlying these associations are mostly unknown. We identified a multi-allelic T-rich sequence (TRS) in the 3'UTR of ATP1B1 that varies in length and sequence composition (T22-27 and T12GT 3GT6). The 3'UTR of ATP1B1 contains 2 functional polyadenylation signals and the TRS is downstream of the proximal polyadenylation site (A2). Therefore, we hypothesized that alleles of this TRS might influence ATP1B1 expression by regulating alternative polyadenylation. In vitro, the T12GT 3GT6 allele increases polyadenylation at the A2 polyadenylation site as compared to the T23 allele. Consistent with our hypothesis, the relative abundance of the A2-polyadenylated ATP1B1 mRNA was higher in human kidneys with at least one copy of the T12GT 3GT6 allele than in those lacking this allele. The T12GT 3GT6 allele is also associated with higher systolic BP (beta = 3.3 mmHg, p = 0.014) and diastolic BP (beta = 2.4 mmHg, p = 0.003) in a European-American population. Therefore, we have identified a novel multi-allelic TRS in the 3'UTR of ATP1B1 that is associated with higher BP and may mediate its effect by regulating the polyadenylation of the ATP1B1 mRNA.