Dual PI-3 kinase/mTOR inhibition impairs autophagy flux and induces cell death independent of apoptosis and necroptosis

The PI-3 kinase (PI-3K)/mTOR pathway is critical for cell growth and proliferation. Strategies of antagonising this signaling have proven to be detrimental to cell survival. This observation, coupled with the fact many tumours show enhanced growth signaling, has caused dual inhibitors of PI-3K and mTOR to be implicated in cancer treatment, and have thus been studied across various tumour models. Since PI-3K (class-I)/mTOR pathway negatively regulates autophagy, dual inhibitors of PI-3K/mTOR are currently believed to be autophagy activators. However, our present data show that the dual PI-3K/mTOR inhibition (DKI) potently suppresses autophagic flux. We further confirm that inhibition of Vps34/PI3KC3, the class-III PI-3K, causes the blockade to autophagosome-lysosome fusion. Our data suggest that DKI induces cell death independently of apoptosis and necroptosis, whereas autophagy perturbation by DKI may contribute to cell death. Given that autophagy is critical in cellular homeostasis, our study not only clarifies the role of a dual PI-3K/mTOR inhibitor in autophagy, but also suggests that its autophagy inhibition needs to be considered if such an agent is used in cancer chemotherapy

LCK, survivin and PI-3K in the molecular biomarker profiling of oral lichen planus and oral squamous cell carcinoma.

T cell signaling is critical in oral lichen planus (OLP) based on the pathogenesis of this chronic inflammatory autoimmune mucocutaneous lesion. Lck plays a key role in T cell signaling; ultimately this signaling affects other targets such as PI-3K. Excessive activity in PI-3K inhibits apoptosis and promotes uncontrolled cell growth. Molecular biomarker profiling in OLP, Chronic Interface Mucosities (CIM), Epithelial Dysplasia (EpD) and Oral Squamous Cell Carcinoma (SCCA) with application of the principle of biomarker voting may represent a new frontier in the diagnosis, assessment and the arguable debate of OLP transformation to cancer. The presence of Lck, PI-3K and Survivin, a cancer specific anti-apoptotic protein was assessed, using immunohistochemistry and tissue micro-array on patient samples, in OLP, SCCA, CIM and EpD. Lck expression was very high in 78.6 % of OLP patients compared to 3.7% in SCCA; PI-3K was high in 63% of SCCA, 100% of EpD, and 35.7% OLP cases. Survivin was high in 64.3% of OLP cases, 96.3% of SCCA, and 100% of EpD. CIM cases may be slightly different molecularly to OLP. Taken together, our data suggest that biomarker protein voting can be effectively used to isolate high-risk OLP cases. Specifically, we show data with four remarkable cases demonstrating that molecular factors are predictive of histopathology. We conclude that it is safer to treat OLP as premalignant lesions, to adopt aggressive treatment measure in histopathologic described well and moderately differentiated SCCA, and to monitor progress of these diseases molecularly using individualized auto-proteomic approach. The use of Lck inhibitors in OLP management needs to be investigated in the future

New series involving binomial coefficients

In this paper, we deduce several new identities on infinite series with denominators of summands containing both binomial coefficients and linear parts. We mainly evaluate the sums $$\sum_{k=1}^\infty\frac{x_0^k}{(2k-1)\binom{3k}k},\ \sum_{k=0}^\infty\frac{x_0^k}{(3k+1)\binom{3k}k} \ \text{and}\ \sum_{k=0}^\infty\frac{x_0^k}{(3k+2)\binom{3k}k}$$ for any $x_0\in(-27/4,27/4)$. In particular, we have $$\sum_{k=1}^\infty\frac{(\frac{9-3\sqrt3}2)^k}{(2k-1)\binom{3k}k}=(\sqrt3-1)\log(\sqrt3+1).$$ We also prove that $$\sum_{k=1}^\infty \frac{\sum_{k<j\le 3k}1/j}{(2k-1)2^k\binom{3k}k}=\frac{\pi^2}{36}-\frac{\pi}{18} -\frac2{15}(G+\log^22)+\frac{\log2}3,$$ where $G$ is the Catalan constant. In addition, we pose some new conjectural series identities involving binomial coefficients; for example, we conjecture that $$\sum_{k=1}^\infty\frac{\binom{2k}k^3}{4096^k}\bigg(9(42k+5)\sum_{j=0}^{k-1}\frac1{(2j+1)^4}+\frac{25}{(2k+1)^3}\bigg)=\frac 56\pi^3.$$Comment: 19 page

PI-3K and Akt are mediators of AP-1 induction by 5-MCDE in mouse epidermal Cl41 cells

5-Methylchrysene has been found to be a complete carcinogen in laboratory animals. However, the tumor promotion effects of (±)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) remain unclear. In the present work, we found that 5-MCDE induced marked activator protein-1 (AP-1) activation in Cl41 cells. 5-MCDE also induced a marked activation of phosphatidylinositol 3-kinase (PI-3K). Inhibition of PI-3K impaired 5-MCDE–induced AP-1 transactivation, suggesting that PI-3K is an upstream kinase involved in AP-1 activation by 5-MCDE. Furthermore, we found that Akt is a PI-3K downstream mediator for 5-MCDE–induced AP-1 transactivation, whereas another PI-3K downstream kinase, p70S6K, was not involved in AP-1 activation by 5-MCDE. Moreover, inhibition of Akt activation blocked 5-MCDE–induced activation of extracellular signal–regulated protein kinases (ERKs) and c-Jun NH2-terminal kinases (JNKs), whereas it did not affect p38K activation. Consistently, overexpression of a dominant-negative mutant of ERK2 or JNK1 blocked the AP-1 activation by 5-MCDE. These results demonstrate that 5-MCDE is able to induce AP-1 activation, and the AP-1 induction is specifically through a PI-3K/Akt–dependent and p70S6K-independent pathway

Involvement of JNK-mediated pathway in EGF-mediated protection against paclitaxel-induced apoptosis in SiHa human cervical cancer cells

We investigated the signalling pathways by which epidermal growth factor (EGF) modulates paclitaxel-induced apoptosis in SiHa human cervical cancer cells. SiHa cells exposed to paclitaxel underwent apoptosis, which was strongly inhibited by EGF. This inhibition of apoptosis by EGF was not altered by pharmacological blockade of phosphatidylinositol 3′-OH kinase (PI-3K) with the PI-3K specific inhibitor LY294002 or blockade of the mitogen-activated protein kinase (MAPK) kinase (MEK) with the MEK specific inhibitor PD98059, or by transfection of the cells with PI-3K or MEK dominant-negative expression vectors. EGF did not stimulate PI-3K/Akt, MEK/MAPK, or p38 MAPK activity in SiHa cells but did transiently activate the c-Jun NH2-terminal kinase (JNK). Co-exposure of SiHa cells to SB202190 at concentrations that inhibit JNK abolished the protective effect of EGF on SiHa cells against paclitaxel-induced apoptosis. Our findings indicate that the JNK signaling pathway plays an important role in EGF-mediated protection from paclitaxel-induced apoptosis in SiHa cells. © 2001 Cancer Research Campaign http://www.bjcancer.co

Super congruences and Euler numbers

Let $p>3$ be a prime. We prove that $$\sum_{k=0}^{p-1}\binom{2k}{k}/2^k=(-1)^{(p-1)/2}-p^2E_{p-3} (mod p^3),$$ $$\sum_{k=1}^{(p-1)/2}\binom{2k}{k}/k=(-1)^{(p+1)/2}8/3*pE_{p-3} (mod p^2),$$ $$\sum_{k=0}^{(p-1)/2}\binom{2k}{k}^2/16^k=(-1)^{(p-1)/2}+p^2E_{p-3} (mod p^3)$$, where E_0,E_1,E_2,... are Euler numbers. Our new approach is of combinatorial nature. We also formulate many conjectures concerning super congruences and relate most of them to Euler numbers or Bernoulli numbers. Motivated by our investigation of super congruences, we also raise a conjecture on 7 new series for $\pi^2$, $\pi^{-2}$ and the constant $K:=\sum_{k>0}(k/3)/k^2$ (with (-) the Jacobi symbol), two of which are $$\sum_{k=1}^\infty(10k-3)8^k/(k^3\binom{2k}{k}^2\binom{3k}{k})=\pi^2/2$$ and $$\sum_{k>0}(15k-4)(-27)^{k-1}/(k^3\binom{2k}{k}^2\binom{3k}k)=K.$

Molecular Mechanism of Tetraspanin CD9 Mediated Cell Motility

CD9, a member of the tetraspanin superfamily of proteins participates in the regulation of cell adhesive functions such as cell migration. The mechanisms underlying CD9 mediated cell migration are not known. In the current study, we investigated the molecular basis for the CD9 promoted cell migration. Our findings show that the phosphatidylinositol-3 kinase (PI-3K) inhibitors, wortamannin and LY294002 inhibited CD9 promoted cell motility in Chinese hamster ovary (CHO) cells. In contrast, inhibitors targeting protein kinase C or mitogen-activated protein kinase had no effect on CD9 driven CHO cell motility. Furthermore, inhibition of PI-3K activity in CHO cells by dominant/negative PI-3K cDNA transfection abolished CD9 mediated pro-migratory effects. Consistent with these observations, CD9 expression in CHO cells and in the rat aortic smooth muscle (RASM) cells induced enhanced phosphorylation of PI-3K substrate, Akt. In CHO cells, CD9 expression also enhanced protein levels and tyrosine phosphorylation of the adaptor protein p130Cas. However, no significant changes in the CD9 enhanced migration were observed in CHO cells upon down regulation of p130Cas CD9, a member of the tetraspanin superfamily of proteins participates in the regulation of cell adhesive functions such as cell migration. The mechanisms underlying CD9 mediated cell migration are not known. In the current study, we investigated the molecular basis for the CD9 promoted cell migration. Our findings show that the phosphatidylinositol-3 kinase (PI-3K) inhibitors, wortamannin and LY294002 inhibited CD9 promoted cell motility in Chinese hamster ovary (CHO) cells. In contrast, inhibitors targeting protein kinase C or mitogen-activated protein kinase had no effect on CD9 driven CHO cell motility. Furthermore, inhibition of PI-3K activity in CHO cells by dominant/negative PI-3K cDNA transfection abolished CD9 mediated pro-migratory effects. Consistent with these observations, CD9 expression in CHO cells and in the rat aortic smooth muscle (RASM) cells induced enhanced phosphorylation of PI-3K substrate, Akt. In CHO cells, CD9 expression also enhanced protein levels and tyrosine phosphorylation of the adaptor protein p130Cas. However, no significant changes in the CD9 enhanced migration were observed in CHO cells upon down regulation of p130Cas by siRNA transfection suggesting that p130Cas dependent pathways are not mandatory for CD9 mediated motility. To further understand the mechanisms by which CD9 regulates cell migration, we studied the relative contribution of the fibronectin (FN) receptor integrin, alpha-5-beta-1 in CD9 mediated cell motility. Our findings show that CD9 is in molecular complex with alpha-5-beta-1 and that CD9 promoted migration can be completely blocked by an alpha-5-beta-1 function blocking antibody. Further studies revealed that CD9 expression may stabilize the active conformer of the beta-1 integrin. Taken togeather, our study demonstrates key molecular mechanisms governing CD9 mediated haptotactic cell motility to FN in CHO cells. Our findings indicate that CD9 in concert with integrin alpha-5-beta-1 requires activation of the PI-3K pathway leading to enhanced haptotactic cell migration on FN