Hari Kotturi

Conducting scaffolds for liver tissue engineering

It is known that there is a correlation between a cell membrane potential and the proliferation of the cell. The high proliferation capacity of liver cells can also be attributed to its specific cell membrane potential as liver cell is recognized as one of the most depolarized of all differentiated cells. We hypothesized that this phenomenon can be emphasized by growing liver cells in conducting scaffolds that can increase the electrical communication among the cells. In this article, using tissue engineering techniques, we grew hepatocyte cells in scaffolds with various compositions. It was found that the scaffolds containing conducting polymer of poly (3,4-ethylenedioxythiophene) (PEDOT) provide the best condition for attachment and proliferation of the cells. More specifically, the blend of hyaluronan, PEDOT, and collagen (I) as dopants in gelatin-chitosan-based scaffold presented the best cell/scaffold interactions for regeneration of liver cells.

Mechanical properties of natural chitosan/hydroxyapatite/magnetite nanocomposites for tissue engineering applications

Chitosan (CS), hydroxyapatite (HA), and magnetite (Fe3O4) have been broadly employed for bone treatment applications. Having a hybrid biomaterial composed of the aforementioned constituents not only accumulates the useful characteristics of each component, but also provides outstanding composite properties. In the present research, mechanical properties of pure CS, CS/HA, CS/HA/magnetite, and CS/magnetite were evaluated by the measurements of bending strength, elastic modulus, compressive strength and hardness values. Moreover, the morphology of the bending fracture surfaces were characterized using a scanning electron microscope (SEM) and an image analyzer. Studies were also conducted to examine the biological response of the human Mesenchymal Stem Cells (hMSCs) on different composites. We conclude that, although all of these composites possess in-vitro biocompatibility, adding hydroxyapatite and magnetite to the chitosan matrix can noticeably enhance the mechanical properties of the pure chitosan.

Tumor cells expressing a fusion protein of MULT1 and Fas are rejected in vivo by apoptosis and NK cell activation

Tumor cells evade immunosurveillance by elements of the innate immune system, such as natural killer (NK) cells, by downregulating or ‘shedding’ certain cell-surface molecules like mouse UL16-binding protein-like transcript 1 (MULT1) that can activate NK cells through NK cell receptors such as NKG2D; they also avoid Fas-mediated apoptosis by downregulating its expression. In the present study we report the design and evaluation of the antitumor activity of a novel fusion protein, MULT1E/FasTI, consisting of the extracellular domain of MULT1 and the transmembrane and intracellular domains of Fas. The fusion construct (pMULT1E/FasTI) was transfected into the mouse pulmonary carcinoma cell line TC-1; and stable cell clones expressing the fusion protein were established. In-vitro cell culture studies demonstrated that the binding of the NKG2D/Fc, a recombinant protein of mouse NK cell receptor, to MULT1E/FasTI expressed on tumor cells was able to elicit apoptosis as assayed by Annexin V–fluorescein isothiocyanate staining and caspase-3 enzyme-linked immunosorbent assay and to activate NKG2D-expressing cells, such as NK cells. In-vivo subcutaneous tumor studies demonstrated that tumor cells expressing MULT1E/FasTI grew significantly slower than cells without the protein. Pulmonary metastasis studies showed that most of the mice completely rejected tumor cells expressing MULT1E/FasTI. This approach may generate a new therapeutic agent for tumor treatment when combined with tumor cell-specific gene delivery vehicles such as oncolytic adenovirus vectors.

Genomic Sequence of Mycobacteriophage OKCentral2016

ABSTRACT OKCentral2016 is the first mycobacteriophage sequenced from Oklahoma soil using the bacterial host Mycobacterium smegmatis strain mc2155. OKCentral2016 has a double-stranded DNA genome composed of 50,072 bp, with 84 predicted coding genes and 1 tRNA sequence. This mycobacteriophage has sequence similarities to members of the A10 subcluster.

Abstract A39: 3,4',5- Trismethoxybenzophenone inhibits the growth of human hepatocarcinoma cells via cell-cycle arrest at the G2/M phase

3,49,5-trismethoxybenzophenone (TMBP) is an analogue of the natural dietary polyphenolic compound resveratrol (3,5,49-trihydro-trans-stilbene, abbreviated as RVT). Natural RVT belongs to the phytoalexin class of phytochemicals that are antibiotics produced by plants in response to stimuli such as injury, ultraviolet irradiation, stress, and fungal attack. RVT and its analogues have also been shown to prevent hepatic steatosis. In this study we focused on TMBP which is a methylated derivative of RVT. Studies have shown that TMBP exhibits added biological effects and a better pharmacokinetic profile compared to RVT due to the presence of methoxy groups. The human hepatoma cell line (FCA4 cells) that harbors a subgenomic selectable HCV replicon was used for this study. The Huh7 cell line lacking the replicon served as the corresponding control. MTS assay was used to determine the cytotoxicity of TMBP at various concentrations. Wound healing assay was performed to determine the effect of the analogue on cell migration. Flow cytometry and magnetic levitation were used to measure the effect of compound on cell cycle and 3D spheroid formation. Our data showed that treating liver cancer cells with TMBP resulted in a significant dose and time dependent growth inhibition combined with G2/M-phase cell cycle arrest at 5 μM with (IC50) at 7.5 μM. TMBP at 5μM inhibited the formation of 3D spheroids, and also repressed the migration of cancer cells. Current therapies available for treating hepatocellular carcinoma (HCC) have a high incidence of recurrence and postoperative death in patients. Exploring different strategies with high pharmaceutical function and low toxicity is of great urgency for treating HCC. Our studies show that we have identified a promising analogue for treating HCC. Citation Format: Christopher J. Patton, Pritika Khadka, Hari Kotturi. 3,49,5- Trismethoxybenzophenone inhibits the growth of human hepatocarcinoma cells via cell-cycle arrest at the G2/M phase. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A39.

Abstract 4552: Trimethoxy-cis-stilbene exhibits potent anti-tumor activities via suppression of AKT signaling and cell cycle arrest in virus-induced hepatocellular carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world, and the incidence is likely to increase due to other health complications such as obesity and diabetes. Chronic hepatitis C virus (HCV) infection and cirrhosis are considered to be major risk factors for induction of HCC. HCV induces cirrhosis, regenerative nodules and cancer stem cell (CSC)-related proteins in the liver. Treatments for chronic hepatitis C and HCC have been challenging so far. Therefore, identification of novel therapeutic agents that target HCV and/or HCV-induced HCC is an unmet medical need. Resveratrol (RES) and its synthetic analogue, trimethoxy-cis-stilbene (TMS) have shown to possess a range of therapeutic activities against various diseases. Although RES has been extensively studied, the anti-viral and anti-tumor activities of TMS have not been fully explored. Methods: HCV subgenomic replicon-expressing hepatoma cells were treated with varying concentrations of RES, TMS or DMSO (control) for 48 hrs. The IC50 values for these drugs were determined based on HCV expression levels in the treated and untreated cultures. Effects of the drugs on cell cycle and AKT signaling pathways were investigated using flow cytometry and protein expression profiles. Cell cytotoxicity of the drugs was determined by MTS assay using normal human hepatocytes (NHH) and hepatoma cells. Magnetic levitation method was carried out to determine effects of the drugs on 3D spheroid cultures. Results: Both RES and TMS downregulated the HCV RNA and NS5B polymerase levels within 48 hr and showed IC50 values equivalent to 100 μM and 1.0 μM, respectively. However, the NHH viability was not compromised during these culture conditions as determined by cytotoxic assays. The anti-HCV effects were accompanied by cell cycle arrest at G2/M phase for TMS, whereas G1/S arrest was observed for RES. TMS-induced G2/M arrest was confirmed by a sharp reduction in phosphorylated CDK1 level in the treated cells. Further analysis showed that active serine/threonine kinase AKT (phosphorylated at Ser473) level was significantly reduced by 1.0 μM TMS. Conversely, the level of p21(Cip1/Waf1), a CDK1 inhibitor, was increased following TMS treatment. Only TMS, but not DMSO (control), successfully inhibited 3D hepatoma spheroid growth in culture. Conclusion: Trimethoxy-cis-stilbene appears to be more potent than its parent compound resveratrol as an anti-HCV and anti-tumor drug in culture conditions. Its anti-tumor effects on HCV-expressing hepatoma cells are exerted by suppression of AKT signaling pathway and cell cycle arrest. This is also supported by an increase in p21(Cip1/Waf1) levels following TMS treatment. Based on these observations, TMS appears to be a promising therapeutic agent for HCV-induced liver diseases including HCC. Citation Format: Charles Nguyen, Hari Kotturi, Sripathi Sureban, Randal J. May, Parthasarathy Chandrakesan, Nathaniel Weygant, Dongfeng Qu, Courtney Houchen, Naushad Ali. Trimethoxy-cis-stilbene exhibits potent anti-tumor activities via suppression of AKT signaling and cell cycle arrest in virus-induced hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4552. doi:10.1158/1538-7445.AM2014-4552