Potential role of vitamin K2 as a chemopreventive agent against hepatocellular carcinoma

Vitamin K, a cofactor necessary for the production of several antihemorrhagic factors, can inhibit the growth of various types of cells derived from neoplasms. In hepatoma cells, vitamin K₂ causes cell-cycle arrest and apoptosis. Vitamin K₂ is widely used in Japan to treat osteoporosis. The safety, relatively low cost and ease of use of vitamin K₂ have led to good compliance with treatment. The result of preliminary clinical trials in patients with chronic liver diseases are intriguing and suggest that vitamin K₂ might reduce the risk of hepatocellular carcinoma (HCC) in patients with liver cirrhosis as well as prevent disease recurrence after curative therapy in patients with HCC. This article reviews the potential role of vitamin K₂ as a chemopreventive agent against HCC and discusses future directions for clinical trials.     

Pharmacological reactivation of p53 as a strategy to treat cancer

It has been confirmed through studies using the technique of unbiased sequencing that the TP53 tumour suppressor is the most frequently inactivated gene in cancer. This finding, together with results from earlier studies, provides compelling evidence for the idea that p53 ablation is required for the development and maintenance of tumours. Genetic reconstitution of the function of p53 leads to the suppression of established tumours as shown in mouse models. This strongly supports the notion that p53 reactivation by small molecules could provide an efficient strategy to treat cancer. In this review, we summarize recent advances in the development of small molecules that restore the function of mutant p53 by different mechanisms, including stabilization of its folding by Apr‐246, which is currently being tested in a Phase II clinical trial. We discuss several classes of compounds that reactivate wild‐type p53, such as Mdm2 inhibitors, which are currently undergoing clinical testing, MdmX inhibitors and molecules targeting factors upstream of Mdm2/X or p53 itself. Finally, we consider the clinical applications of compounds targeting p53 and the p53 pathway.     

磷脂酰肌醇3激酶/蛋白激酶B/核转录因子E2相关因子2信号通路对高糖状态下人甲状腺乳头状癌K1细胞增殖的作用及机制

目的研究核转录因子E2相关因子2(Nrf2)及磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路对高糖状态下人甲状腺乳头状癌K1细胞增殖的影响,并探讨糖代谢相关酶在Nrf2及PI3K/Akt影响K1细胞增殖中的作用。方法根据不同处理因素将K1细胞分为5组:正糖组(正糖5.5 mmol/L培养48 h)、高糖组(正糖5.5 mmol/L培养24 h后,换25 mmol/L高糖继续培养24 h)、Nrf2siRNA组(正糖条件下siRNA转染细胞24 h后,换高糖继续培养24 h)、NcsiRNA组(正糖条件下阴性转染细胞24 h后,换高糖继续培养24 h)、LY294002组(正糖条件下培养24 h后,加50μmol/L LY294002预孵育0.5 h再换用高糖培养24 h)。K1细胞按照上述分组处理后分别用以下方法检测相关指标:MTT法检测细胞增殖,细胞免疫荧光法检测Nrf2蛋白的分布,Western blot检测PI3K、p-PI3K、Akt、p-Akt、核Nrf2、浆Nrf2、葡萄糖-6-磷酸脱氢酶(G6PD)、丙酮酸激酶M2(PKM2)蛋白的表达水平。两组数据比较采用独立样本t检验,多组数据比较采用单因素方差分析。结果与正糖组比较,高糖组K1细胞增殖率、PI3K、p-Akt/Akt、Nrf2、G6PD、PKM2表达显著升高[分别为(87.31±3.67)%比(126.64±5.41)%、0.272±0.039比0.425±0.019、0.168±0.035比0.446±0.021、0.308±0.026比0.597±0.014、0.421±0.024比0.626±0.026、0.198±0.023比0.314±0.023,t=6.109~16.951,均P<0.05],Nrf2在细胞核分布的比例显著升高[(21.6±4.5)%比(91.2±3.5)%,χ2=98.497,P<0.01];与高糖组比较,Nrf2siRNA组K1细胞增殖率明显下降,G6PD、PKM2的蛋白表达明显下调,差异有统计学意义(t=8.936、7.056、8.843,均P<0.01),LY294002组也呈现相似的趋势(t=7.228、6.351、7.910;均P<0.01);与高糖组比较,LY294002组K1细胞PI3K、Akt蛋白水平无明显改变,而p-PI3K、p-Akt、核Nrf2、浆Nrf2蛋白水平及Nrf2核浆蛋白比值均被显著抑制(t=5.748~23.572,均P<0.01)。结论高糖可激活PI3K/Akt信号通路,上调Nrf2表达与核转位,上调磷酸戊糖和糖酵解途径相关酶PKM2、G6PD的表达,促进K1细胞增殖。     

Activation of Rictor/mTORC2 signaling acts as a pivotal strategy to protect against sensorineural hearing loss

The Food and Drug Administration–approved drug sirolimus, which inhibits mechanistic target of rapamycin (mTOR), is the leading candidate for targeting aging in rodents and humans. We previously demonstrated that sirolimus could treat ARHL in mice. In this study, we further demonstrate that sirolimus protects mice against cocaine-induced hearing loss. However, using efficacy and safety tests, we discovered that mice developed substantial hearing loss when administered high doses of sirolimus. Using pharmacological and genetic interventions in murine models, we demonstrate that the inactivation of mTORC2 is the major driver underlying hearing loss. Mechanistically, mTORC2 exerts its effects primarily through phosphorylating in the AKT/PKB signaling pathway, and ablation of P53 activity greatly attenuated the severity of the hearing phenotype in mTORC2-deficient mice. We also found that the selective activation of mTORC2 could protect mice from acoustic trauma and cisplatin-induced ototoxicity. Thus, in this study, we discover a function of mTORC2 and suggest that its therapeutic activation could represent a potentially effective and promising strategy to prevent sensorineural hearing loss. More importantly, we elucidate the side effects of sirolimus and provide an evaluation criterion for the rational use of this drug in a clinical setting.

With increasing progress and a rapid improvement in the living standards of individuals in society, considerable importance is being given to hearing problems. Therefore, it is of great significance to study the occurrence and understand the mechanisms underlying the development of hearing disorders to effectively prevent, detect, and treat deafness. Among the hearing-impaired population, sensorineural deafness accounts for the majority of cases of hearing loss. Studies have shown that almost 50% of hereditary sensorineural deafness is caused by mutations in hair cell (HC) enriched genes, although HCs constitute only approximately one-tenth of the cells in the sensory epithelium of the cochlea (1). These factors responsible for injury, including genetic defects, can lead to irreversible damage and the loss of cochlear HCs, which is the main cause of sensorineural hearing loss (2). Therefore, understanding the mechanism of the development and survival of auditory HCs and being able to restore the function of cochlear sensory epithelium is an ideal approach in auditory reconstruction and also the primary focus in the field of otology.Mechanistic target of rapamycin (mTOR), now defined as the mechanistic target of sirolimus, is a highly conserved serine/threonine protein kinase, which controls the growth of cells and organisms induced by growth factors and nutrients. The mTOR is assembled into two multiprotein complexes, namely, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which can be distinguished based on their related proteins and their sensitivity to sirolimus (3). Evidence indicates that mTORC1 is mainly responsible for cell growth and proliferation in response to growth factors, nutrients, or stress, and that the two main downstream targets of mTORC1, namely, p70S6 kinase (S6K) and elongation factor 4E binding protein (4E-BP1), are key regulators of cap-dependent protein translation (4, 5). Although the mechanisms that regulate mTORC1 are well understood, the aspect of regulation of mTORC2 is relatively poorly characterized. The mTORC2 signaling is insensitive to nutrients; however, it responds to growth factors, such as insulin, through poorly defined mechanism(s) that require(s) PI3K (6). Experiments in yeast and cultured mammalian cells have indicated that mTORC2 plays a role in the regulation of the actin cytoskeleton (7, 8). The mTORC2 controls several members of the AGC kinase subfamily, including Akt, protein kinase C-α (PKC-α), and serum and glucocorticoid-induced protein kinase 1 (SGK1) (9–11). Although mTORC2 is relatively insensitive to acute sirolimus treatment, recent studies have shown that prolonged exposure to sirolimus can inhibit the mTORC2 complex assembly (12–14).As one of the central regulators of cellular activities, the mTOR signaling pathway has been attracting increasing attention in recent years (4, 15). Several studies now focus on the function of mTOR in metabolic tissues, largely because these tissues are particularly sensitive to the three inputs (nutrients, insulin, and energy status) that control mTOR (16, 17). Additionally, abnormalities in the mTOR signaling pathway can lead to conditions such as epilepsy (18, 19), diabetes mellitus (20, 21), tuberous sclerosis syndrome (22, 23), and tumors (24, 25). Nevertheless, existing knowledge regarding the functioning and regulation of the mTOR pathway in the maintenance of the auditory system and homeostasis is limited. To date, the available evidence suggests that auditory disorders result from changes in the mTOR signaling pathway (26–28). Recently, our study group provided evidence that inhibition of mTORC1 resulting from an intraperitoneal (i.p.) injection of 1 mg/kg sirolimus or from a genetic disorder leading to a decrease in mTORC1 activity via deficiency of Raptor attenuated age-related hearing loss (ARHL) in C57BL/6J mice, whereas overactivation of the mTORC1 signaling pathway in the neurosensory cells (NSE) caused premature HC death and progressive hearing loss (29). However, as a Food and Drug Administration (FDA)-approved drug, although an i.p. injection of sirolimus in wild-type (WT) C57BL/6J mice can prevent ARHL, some caveats must be considered when translating this finding to a clinical setting. Accordingly, the efficacy and safety of sirolimus should be comprehensively understood prior to its use in treating ARHL. While studying the effects of different doses of the drug, we serendipitously found that high doses of injected sirolimus could result in a significant loss of hearing in mice. Coincidentally, a recent study reports that the inhibition of mTOR by sirolimus results in dose-dependent damage of auditory HCs cultured ex vivo (30). Collectively, these findings suggest the complex functions of the mTOR signaling pathway in the auditory sensory epithelium, especially in auditory HCs. It is currently unknown whether mTOR acts through mTORC1, through mTORC2, or through the synergistic action of these two complexes in the development and survival of HCs. Neither the genetic nor the pharmacological manipulation of mTOR kinase is adequate to distinguish the role of these two complexes in cochlear HCs. In addition, the correlation between mTORC1 and mTORC2 in cochlear HCs and the downstream molecular regulatory signals remains to be elucidated.     

Cdcs1, a major colitogenic locus in mice, regulates innate and adaptive immune response to enteric bacterial antigens

BACKGROUND & AIMS: The absence of interleukin 10, a key cytokine in gut homeostasis, causes severe colitis in C3H/HeJBir but not C57BL/6J mice. The major modifier for colitis was mapped on chromosome 3 and designated cytokine deficiency-induced colitis susceptibility 1 (Cdcs1). We developed reciprocal Cdcs1 congenic stocks on both interleukin 10-deficient backgrounds to identify the susceptibility gene and its function. METHODS: C3H/HeJBir congenic for the C57BL/6J-derived Cdcs1 allele and reciprocal C57BL/6J congenic for the C3H/HeJBir allele were analyzed for colitis development. Parental strains were compared by electrophoretic mobility shift assay to assess the candidacy of nuclear factor-kappaB p50 in the Cdcs1 interval. Functional differences were observed in innate and adaptive immune responses of parental and congenic stocks after bacterial ligand exposure in vitro (cytokine release from bone marrow-derived macrophage and dendritic cells) and in vivo (serum cytokines and primed CD4+ T cell proliferation). RESULTS: Cdcs1 was positioned within a minimum 7-megabase interval containing nuclear factor-kappaB p50. C3H/HeJBir colitis was significantly diminished by the C57BL/6J genome in this interval. Conversely, colitis in C57BL/6J was significantly exacerbated by the reciprocal C3H/HeJBir genome. C3H/HeJBir macrophages constitutively expressed higher nuclear factor-kappaB p50. Functional assays showed that C3H/HeJBir showed reduced innate responsiveness both in vivo and in vitro to bacterial ligands but showed increased CD4 T-cell responses compared with C57BL/6J. This differential responsiveness was controlled by the respective allele at Cdcs1. CONCLUSIONS: The colitogenic Cdcs1 allele impairs innate immunity to bacterial products and in turn skews the adaptive immune response toward compensatory hyperresponsiveness and chronic intestinal inflammation.