Chemically-Modified Tetraiodothyroacetic Acid (Tetrac) Induces Cancer Cell Apoptosis and Facilitates Clearance of Apoptotic Debris (Efferocytosis)

Tetraiodothyroaetic acid (tetrac) is a derivative of L-thyroxine with anticancer properties. By multiple molecular mechanisms, tetrac and chemically-modified tetrac induce apoptosis in a variety of human cancer cells in vitro and in xenografts. The anticancer activities of tetrac are initiated at the thyroid hormone analogue receptor on the extracellular domain of plasma membrane integrin αvβ3 (PJ Davis et al., Physiol Rev 101:319-352, 2021). Induction of apoptosis in glioblastoma xenograft with chemically modified tetrac (P-bi-TAT) has yielded 90% in volume of grafts that continues after discontinuation of tetrac. In the present study, we show that human glioblastoma xenograft shrinkage in response to P-bi-TAT is associated with local appearance of phagocytic monocytes and clearance of apoptotic debris (efferocytosis). Primary culture xenograft of glioblastoma cells (GBM 052814, kindly provided by the University of Pittsburgh Medical Center, Department of Neurosurgery) and U87-luc (ATCC, Manassas, VA) xenografts were generated in 5-member groups of nude mice for each tumor cell type and for controls. Five days post-implantation, injection of animals was begun with PBS (control) or P-bi-TAT (10 mg/kg body weight). Injection was continued X21 days and animals were then maintained off-treatment for an additional 21 days. Tumors were harvested, formalin-fixed and slide-mounted, then analyzed by TUNEL assay for apoptosis and by anti-CD68 staining for monocytic macrophage content. Histologic analysis (H&E staining) was also carried out. TUNEL analysis and histopathology of both xenograft models revealed more than 90% apoptotic change with 21-days of P-bi-TAT treatment (P <0.001) and persistence of 40% apoptotic change 3 weeks post-discontinuation of drug (P<0.001 vs. end of treatment change). By H&E histology and CD68 analysis, monocytes accounted for more than 90% of the viable cells after 3 weeks’ drug treatment. Sixty percent of the end-of-treatment monocyte population persisted 3 weeks after discontinuation of P-bi-TAT (P <0.001). Histology revealed negligible cell debris after 3 weeks of drug treatment and at 3 weeks post-discontinuation of P-bi-TAT. Thus, the anticancer/pro-apoptotic action of tetrac-containing P-bi-TAT is associated with efferocytosis that contributes to the frank tumor shrinkage that results from P-bi-TAT treatment of human glioblastoma xenografts. This is the first documentation of efferocytosis regulated from the thyroid hormone analogue receptor on tumor cell integrin αvβ3.

Cytotoxic potentials of silibinin assisted silver nanoparticles on human colorectal HT-29 cancer cells

It is of interest to study the cytotoxicity of silibinin assisted silver nanoparticles in human colorectal (HT-29) cancer cells. Silver nanoparticles were synthesized using silibinin as a reducing agent. The synthesized silibinin assisted silver nanoparticles (SSNPs) were characterized and analyzed using a transmission electron microscope and spectrophotometer. The SSNPs synthesized in this study are spherical and their size ranges from 10 to 80 nm. HT-29 cells were treated with different concentrations (2, 4, 6, 8 and 10 ng/mL) of SSNPs and cytotoxicity was evaluated. The apoptosis was using flow cytometry. p53 protein expression using western blot. SSNPs are induced a decrease in viability and increased concentration-dependent cytotoxicity in HT-29 cells. SSNPs treatment also caused apoptosis-related morphological changes. SSNPs treatments at 8 and 16 ng/ml showed a prominent apoptotic change i.e., 70.3% and 83.6% respectively, and decreased viability of HT-29 cells 20% and 11.2% respectively as compared to control cells. SSNPs treatments induced p53 expression in HT-29 cells. Data shows that SSNPs have the potential to induce apoptosis in colorectal cancer cells. This provides insights for the further evaluation of SSNPs in fighting colon cancer.

Apoptotic Change Is a Major Reason for Defect in Early Erythroid Development in Cell Line Models for Ribosomal Protein (RP) S19 Deficient Diamond-Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein (RP) S19 gene. It is not known how the RPS19 deficiency impairs erythopoiesis and proliferation of hematopoietic progenitors. The majority of cases appear to result from an intrinsic disorder of the erythroid progenitor that involves its inability to respond normally to inducers of erythroid proliferation and differentiation. Erythropoietin (Epo) controls the proliferation, differentiation and survival of the erythroid progenitors. However, so far, no receptor-ligand defects have yet been identified. We have established an in vitro models for RPS19 deficient DBA using lentiviral vector mediated doxycycline (Dox) inducible small interfering RNA (siRNA) against RPS19 (Blood102:504a, 2003). To analyze the effect of suppression of RPS19 for erythroid differentiation, we used cytokine dependent TF-1 cell lines (CD34+, CD71+, GFAlow) together with new established cytokine independent K562 cell lines (CD34−, CD71+, GFAhigh). We established two types of cell lines (TF-1A, K562A and TF1B, K562B) using different siRNA against RPS19. Five days after Dox induction, RPS19 protein level was suppressed to 45–55% in TF-1A and K562A, 65–75% in TF1B and K562B compared to control scramble transduced cell lines (TF1S, K562S) by western blot analysis. Suppression of cell growth and colony formation correlated with the suppression level of RPS 19 in TF1A, B and K562A, B cells. In contrast, after stimulation with Epo, glycophorin A expression, measured by flow cytometry, and hemoglobin content (DAF staining) showed suppression of erythroid differentiation only in TF1A and TF1B but not in K562A and K562B. Since Epo induces the stimulation of Jak2 tyrosine kinase which leads to the tyrosine phosphorylation of several proteins including the Epo receptor itself. As a result, different intracellular pathways are activated such as Ras/MAP kinase, phosphatidylinositol 3-kinase and STAT transcription factors. Therefore, we analyzed Epo stimulated signal transduction in these cell lines. However, no abnormal signal transduction could be detected in any of the cell lines. Cell cycle analysis showed that the percentage of cells in the G0/G1 phase increased and apoptotic cells detected by Annexin-V analysis also increased in TF1 (A<B) and K562 (A<B) cells. Western blot analyzed p21 showed that the level of p21 was increased in TF1 (A<B) and K562 (A<B) cells. These results indicate that Epo triggered onset of terminal maturation is intact in RPS19 deficient DBA cell line models. We speculate that apoptotic change by suppressed RPS19 may be one of the major reason for the accelerated loss of erythroid progenitor clonogenicity in RPS19 deficient DBA. These cell lines are useful to determine the mechanisms of RPS19 deficient DBA.