Sirt3 restricts tumor initiation via promoting LONP1 deacetylation and K63 ubiquitination.

BACKGROUND: Sirtuin 3 (Sirt3) is a controversial regulator of carcinogenesis. It residents in the mitochondria and gradually decays during aging. In this study, we tried to investigate the role of Sirt3 in carcinogenesis and to explore its involvement in metabolic alteration. METHODS: We generated conditional intestinal epithelium Sirt3-knockout mice by crossing ApcMin/+; Villin-Cre with Sirt3fl/fl (AVS) mice. The deacetylation site of Lon protease-1 (LONP1) was identified with Mass spectrometry. The metabolic flux phenotype was determined by Seahorse bioanalyzer. RESULTS: We found that intestinal epithelial cell-specific ablation of Sirt3 promotes primary tumor growth via stabilizing mitochondrial LONP1. Notably, we newly identified that Sirt3 deacetylates human oncogene LONP1 at N terminal residue lysine 145 (K145). The LONP1 hyperacetylation-mutant K145Q enhances oxidative phosphorylation to accelerate tumor growth, whereas the deacetylation-mutant K145R produces calorie-restriction like phenotype to restrain tumorigenesis. Sirt3 deacetylates LONP1 at K145 and subsequently facilitates the ESCRT0 complex sorting and K63-ubiquitination that resulted in the degradation of LONP1. Our results sustain the notion that Sirt3 is a tumor-suppressor to maintain the appropriate ubiquitination and degradation of oncogene LONP1. CONCLUSION: Sirt3 represents a targetable metabolic checkpoint of oncogenesis, which produces energy restriction effects via maintaining LONP1 K145 deacetylation and subsequent K63 ubiquitination.

Modified Shenlingbaizhu Decoction represses the pluripotency of colorectal cancer stem cells by inhibiting TGF-ß mediated EMT program.

BACKGROUND: The Modified Shenlingbaizhu Decoction (MSD) utilizes various phytomedicines has been applied to treat colorectal cancer (CRC). Colorectal cancer stem cells (CSCs) have proven to be tightly associated with CRC progression and metastasis. The mechanism of MSD's inhibitory effect on CSCs has not been determined. PURPOSE: To figure out how MSD inhibits the pluripotency of CSCs and impedes the EMT program. METHODS: The ingredients of MSD extracts were characterized by high-performance liquid chromatography (HPLC). BALB/c-nu mice were transplanted into EGFP labeled SW480 CRC cells and the tumor weight and volume were recorded before and after various doses of MSD treatment. The concentration of TGF-ß1 was quantified with an Enzyme-linked immunosorbent assay. To delineate the logical relationship between EMT and CSCs regulated by MSD, TGF-ß/Smad inhibitor and activator were adopted in tumor-bearing mice and diverse CRC cell lines. Cancer stem cell markers were analyzed by flow cytometry. In vitro analysis of cell motility and viability were done using CCK-8, wound healing, and invasion assay. Immunohistochemistry (IHC) and western blotting (WB) were used for detecting protein expression. The collected results were statistically analyzed with GraphPad Prism 8.0. RESULTS: MSD treatment significantly reduced the size of colorectal cancer tumors and lowered the serum content of TGF-ß1 in mice. Importantly, MSD markedly reduced the expression of pluripotent factors and depressed CD133+ stem cells in the tumor tissues. The TGF-ß/Smad inhibitor neutralized the EMT signaling and lowered the pluripotency by dephosphorylation of SMAD2/3. Similarly, MSD attenuated the pluripotency by limiting TGF-ß/Smad signaling-induced EMT in vivo. MSD inhibited colorectal cancer cell proliferation, migration, and invasion. CONCLUSIONS: MSD inhibits the growth of colorectal cancer. It dampens the pluripotency of CSCs by repressing the TGF-ß-induced EMT program.

Dahuang Fuzi Baijiang decoction restricts progenitor to terminally exhausted T cell differentiation in colorectal cancer.

Obesity increases the risk of colorectal cancer (CRC) by 30%. The obese tumor microenvironment compromises antitumor immunity by eliciting exhausted T cells (Tex). Hypothesizing that Dahuang Fuzi Baijiang decoction (DFB) is a combined classical prescription from the "Synopsis of Prescriptions of the Golden Chamber". We first determined that DFB regresses tumor growth in high-fat diet-induced obese mice by expanding the TIM3- subset with intermediate expression of programmed cell death-1 (PD-1int TIM3- ) and restricting the PD-1hi TIM3+ subset. Transcription factor 1 (TCF1) is highly expressed in the PD-1int TIM3- subset but is absent in PD-1hi TIM3+ cells. We next confirmed that progenitor PD-1int TCF+ cells robustly produce tumor necrosis factor-α (TNFα) and interferon-γ, whereas terminally differentiated PD-1int TCF+ cells have defects in generating TNFα. With transgenic ob/ob mice, we found that DFB produces cooperative efficacy with anti-PD-1 (αPD-1) by limiting the PD-1hi Tim3+ subset and amplifying the PD-1int TCF+ population. Finally, we defined the recombinant chemokine C-C-motif receptor 2 (CCR2)+ CD8+ subset as terminal Tex and identified that the differentiation from progenitor to terminal Tex is driven, at least in part, by the chemokine (C-C motif) ligand 2 (CCL2)/CCR2 axis. The CCR2 inhibitor enhances the response to αPD-1 by promoting the counts of progenitor Tex. Altogether, DFB dampens CCL2 and preserves progenitor Tex in the obese microenvironment to restrain CRC progression. These findings provide unambiguous evidence that the traditional Chinese formula DFB can prevent tumor progression by modulating adaptive immunity and establish a strong rationale for further clinical verification.

microRNA-935-modified bone marrow mesenchymal stem cells-derived exosomes enhance osteoblast proliferation and differentiation in osteoporotic rats.

AIMS: The involvement of several microRNAs (miRNAs) in osteogenic differentiation has been indicated recently. Also, exosomes, derived from different cells, could shuttle specific miRNAs to other cell systems. Nevertheless, the effect and mechanism of microRNA-935 (miR-935)-containing exosomes in osteoblasts remain basically unclear. The current work was set to inspect the relevance of bone marrow mesenchymal stem cells (BMSCs)-derived exosomes (BMSC-exo) carrying miR-935 to osteoporotic rats. METHODS: The extracted BMSCs and purchased osteoblasts were cultured, followed by exosome isolation and identification. After cell grouping, osteoblasts were co-cultured with BMSCs. CCK-8, alizarin red staining as well as ALP staining were performed to detect osteoblast proliferation and activity. The binding connection between miR-935 and signal transducer and activator of transcription 1 (STAT1) was measured by dual-luciferase reporter gene assays. The expression profiles of miR-935, STAT1 and osteoblast-related proteins were assessed by RT-qPCR and Western blot. A rat model with osteoporosis was induced, and the BMD, BV/TV, Tb.N, Tb.Th and Tb.Sp values in rat bone tissues were observed by Micro-CT. RESULTS: BMSC-exo inhibited STAT1 levels by the delivery of miR-935 into osteoblasts, while STAT1 silencing promoted ALP activity in osteoblasts and mineralized nodules. STAT1 was identified as a target gene of miR-935. Moreover, in vivo experiments showed that in ovariectomized rats, silencing of miR-935 significantly reduced BMD, BV/TV, Tb.N, Tb.Th and increased Tb.Sp. CONCLUSION: BMSC-exo carry miR-935 to promote osteoblast proliferation and differentiation through targeting STAT1.

"Sclerotic Band" type of classification system and measurement of necrotic area for osteonecrosis of the femoral head.

Osteonecrosis of the femoral head is a common orthopedic disease. Based on years of clinical experience and significant imaging data, this study aimed to elucidate a new type of it, to help improve prognosis in young adults and provide a basis for hip preservation treatment.From January 2014 to December 2016, a total of 211 patients undergoing hip preservation surgery for femoral head necrosis at our hospital were enrolled in this study. Coronal plane classification and cross-sectional area analysis were performed by nuclear magnetic resonance imaging (computed tomography optional) in cases meeting the inclusion criteria. Meanwhile, a new method of classification and calculating the necrotic area was proposed. The application simulation was conducted using sample cases. Additionally, treatment methods were recommended. We used our method to compare the outcome of the selected patients with the JIC classification so as to judge the advantages and disadvantages.The " pressure bone trabecular angle " of the femoral head was measured, and the "sclerotic band" (Zhang Ying) type of classification system and the "quartile" (Zhang Ying) method of measurement were used in 2 sample cases. After analysis, it is more accurate than JIC.The "Sclerotic band" type of classification system and 'quartile' methods are new methods to evaluate the stability of femoral head necrosis. They are convenient for clinical application and easily adopted.

LncRNA NKILA integrates RXFP1/AKT and NF-κB signalling to regulate osteogenesis of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are previously found to have potential capacity to differentiate into osteocytes when exposed to specific stimuli. However, the detailed molecular mechanism during this progress remains largely unknown. In the current study, we characterized the lncRNA NKILA as a crucial positive regulator for osteogenesis of MSCs. NKILA attenuation significantly inhibits the calcium deposition and alkaline phosphatase activity of MSCs. More interestingly, we defined that NKILA is functionally involved in the regulation of RXFP1/PI3K-AKT and NF-κB signalling. Knockdown of NKILA dramatically down-regulates the expression of RXFP1 and then reduces the activity of AKT, a downstream regulator of RXFP1 signalling which is widely accepted as an activator of osteogenesis. Moreover, we identify NF-κB as another critical regulator implicated in NKILA-mediated osteogenic differentiation. Inhibition of NF-κB can induce the expression of RUNX2, a master transcription factor of osteogenesis, in a HDAC2-mediated deacetylation manner. Thus, this study illustrates the regulatory function of NKILA in osteogenesis through distinct signalling pathways, therefore providing a new insight into searching for new molecular targets for bone tissue repair and regeneration.

Tubeimoside 1 Acts as a Chemotherapeutic Synergist via Stimulating Macropinocytosis.

Macropinocytosis is a highly conserved endocytic process which characterizes the engulfment of extracellular fluid and its contents into cells via large, heterogeneous vacuoles known as macropinosomes. Tubeimoside-1 (TBM1) is a low toxic triterpenoid saponin extracted from a traditional Chinese herb Bolbostemma paniculatum (Maxim.). TBM1 stimulates a quick accumulation of numerous phase-lucent cytoplasmic vacuoles in multiple colorectal cancer (CRC) cell lines. These vacuoles can be termed as macropinosomes that efficiently engulf lucifer yellow. These vesicles are not overlaps with endocytic organelle tracers, such as ERTracker, LysoTracker and mitoTracker. These vacuoles induced by TBM1 partially incorporate into lysosomes. Transmission electron microscope indicates membrane ruffling to form lamellipodia. Protrusions collapse onto and then fuse back with the plasma membrane to generate these large endocytic vacuoles. Notably, TBM1 efficiently trafficks dextrans into heterotopic xenografts in vivo, thus provide consolidated evidence that the vacuolization can be mainly defined as macropinocytosis. TBM1 downregulates cell viability and increases PI-positive, but not highlighted Hoechst 33342-positive cells. TBM1 induced cell death can be ascribed as methuosis by hyperstimulation of macropinocytosis which can be compromised by amiloride derivative 5-(Nethyl-N-isopropyl). Light chain 3 II is recruited to these vesicles to stimulate macropinocytosis. The cell death and vacuoles can be significantly neutralized by chloroquine, but can not be the inhibited by 3-methyladenine. TBM1 can coordinate with 5-FU to exert toxicity reducing and efficacy enhancing effects in vivo by increasing the uptake of the latter without hepatic injury. In conclusion, TBM1 effectively induces in vitro and in vivo macropinocytosis which can traffick small molecules into CRC cells. It is an attractive drug transporter and can be harnessed as a chemotherapeutic synergist with translational potential.

A new 3D printed titanium metal trabecular bone reconstruction system for early osteonecrosis of the femoral head.

Presently, biomechanical support therapy for the femoral head has become an important approach in the treatment of early osteonecrosis of the femoral head (ONFH). Previous studies have reported that the titanium metal trabecular bone reconstruction systems (TMTBRS) achieved satisfactory clinical results for the treatment of early femoral head necrosis. Electron beam melting technology (EBMT) is an important branch of 3D printing technology, which enables the construction of an interface that is required for support of bone in-growth. However, the effect of TMTBRS created using EBMT for clinical applications for early ONFH is still unknown. At present, there are no reports on this topic worldwide. The purpose of this study was to assess the safety of a new 3D printed TMTBRS implant and to evaluate its clinical efficacy in early ONFH.Thirty patients who underwent surgery for ONFH were selected. The stages of ONFH were classified according to the Association Research Circulation Osseus (ARCO) classification. They were followed-up and radiological examination was performed at 6, 12, and 24 months post-surgery to assess TMTBRS stability and bone growth in the bone trabecular holder portion surface. To evaluate hip function, postoperative Harris and Visual Analogue Scale (VAS) scores were used.The postoperative Harris score increased significantly and VAS score decreased significantly at the 12-month follow-up compared to the 24-month follow-up, wherein the Harris score declined slightly and the VAS score was slightly elevated with the aggravation of ONFH. With the passage of time, postoperative improvement rates were 100% for IIA, 70% for IIB, and 0% for IIC. Hip-preserving rates were 100% for IIA, 100% for IIB, and 50% for IIC.The effect of TMTBRS treatment for early ONFH in ARCO IIA and ARCO IIB is satisfactory. However, it is not recommended for a relatively large area of necrosis such as in ARCO IIC.

Effect of blood biochemical factors on nontraumatic necrosis of the femoral head : Logistic regression analysis.

OBJECTIVE: This case-control study aimed to identify the risk factors of nontraumatic necrosis of the femoral head (NONFH). METHODS: In all, 242 patients with NONFH treated at the hip disease research center of our hospital between March 2012 and October 2015 were included. After excluding 19 patients with tumor or tuberculosis, 223 patients were enrolled. Controls comprised 223 healthy persons selected from our hospital database. Single-factor variance analysis and t test were performed to select the index of statistical significance. The 95% confidence interval (95% CI) and normal range of the selected indicators were compared, and abnormal related indexes were selected from the femoral head necrosis group. The selected indicators were based on the increase or decrease to locate the risk indicators and render their corresponding assignment. Logistic regression analysis of the risk factors was performed after the assignment. RESULTS: The necrotic group of patients with decreased carbon dioxide combining power (CO2CP), increased total cholesterol, increased low-density lipoprotein, and decreased high-density lipoprotein levels had statistically significant partial regression coefficient values and the odds ratios were 73.5 (95% CI 24.59-219.74), 7.15 (3.51-14.85), 633.07 (121.7-3304.78), and 20.11 (9.36-43.8), respectively, indicating that these are strong risk factors for NONFH. CONCLUSIONS: Abnormal lipid metabolism is a strong risk factor of NONFH. Lipid examination can be used as a screening tool for NONFH in high-risk populations, for alcoholism, and many hormone applications. The decreased CO2CP was associated with NONFH, and bone microcirculation was considered to possibly lead various conditions such as ischemia and hypoxia-related bone metabolic acidosis. However, further study is needed.