Amphipathic Small Molecule AZT Compound Displays Potent Inhibitory Effects in Cancer Cell Proliferation

Cancer has been identified as a leading cause of death worldwide, and the increasing number of cancer cases threatens to shorten the average life expectancy of people. Recently, we reported a 3-azido-3-deoxythymidine (AZT)-based amphipathic small molecule, ADG-2e that revealed a notable potency against tumor metastasis. To evaluate the anticancer potential of ADG-2e, we assessed its anticancer potency in vitro and in vivo. Anticancer screening of ADG-2e against cervical cancer cells, HeLa CCL2, and BT549 mammary gland ductal carcinoma showed significant inhibition of cancer cell proliferation. Furthermore, mechanistic investigations revealed that cancer cell death presumably proceeded through an oncosis mechanistic pathway because ADG-2e treated cells showed severe damage on the plasma membrane, a loss of membrane integrity, and leakage of α-tubulin and β-actin. Finally, evaluation of the antitumorigenic potential of ADG-2e in mouse xenograft models revealed that this compound potentially inhibits cancer cell proliferation. Collectively, these findings suggest that ADG-2e can evolve as an anticancer agent, which may represent a model for nucleoside-based small molecule anticancer drug discovery

<em>Crif1</em> Deficiency Reduces Adipose OXPHOS Capacity and Triggers Inflammation and Insulin Resistance in Mice

<div><p>Impaired mitochondrial oxidative phosphorylation (OXPHOS) has been proposed as an etiological mechanism underlying insulin resistance. However, the initiating organ of OXPHOS dysfunction during the development of systemic insulin resistance has yet to be identified. To determine whether adipose OXPHOS deficiency plays an etiological role in systemic insulin resistance, the metabolic phenotype of mice with OXPHOS–deficient adipose tissue was examined. Crif1 is a protein required for the intramitochondrial production of mtDNA–encoded OXPHOS subunits; therefore, <i>Crif1</i> haploinsufficient deficiency in mice results in a mild, but specific, failure of OXPHOS capacity <i>in vivo</i>. Although adipose-specific <i>Crif1</i>-haploinsufficient mice showed normal growth and development, they became insulin-resistant. <i>Crif1</i>-silenced adipocytes showed higher expression of chemokines, the expression of which is dependent upon stress kinases and antioxidant. Accordingly, examination of adipose tissue from <i>Crif1</i>-haploinsufficient mice revealed increased secretion of MCP1 and TNFα, as well as marked infiltration by macrophages. These findings indicate that the OXPHOS status of adipose tissue determines its metabolic and inflammatory responses, and may cause systemic inflammation and insulin resistance.</p> </div

Dysregulation of chemokines and activation of stress kinases in <i>Crif1</i>-deficient 3T3-L1 adipocytes.

<p>(A) Silencing of <i>Crif1</i> and expression of adipogenic genes, <i>Ppargamma, adiponectin</i> and <i>Cd36</i> in 3T3-L1 cells. Western blot analysis with anti-ND1 and NDUFA9 (Complex I), anti-UQCRC2 (Complex III) and anti-ATP5A1 (Complex V) after 4 days of <i>Crif1</i> silencing (<i>n</i> = 10). siCTR, control siRNA; si<i>Crif1</i>, <i>Crif1</i> siRNA. Values are means + SD, *p<0.05. (B) Expression of macrophage recruiting chemokines (<i>Mcp1</i>, monocyte attractant chemokine; <i>Ip10</i>, IFN-γ–inducible protein 10; <i>Rantes</i>, Regulated upon Activation, Normal T cell Expressed and Secreted) by 3T3-L1 cells subjected to <i>Crif1</i> siRNA (<i>n</i> = 10). Values are means + SD, *p<0.05. (C) Measurement of MitoSox- or DHE-stained cells by flow cytometry. DHE, di-hydroethidium (<i>n</i> = 10). Values are means + SD, *p<0.05. (D) <i>Mcp1, Ip10</i> and <i>Rantes</i> mRNA levels with/without NAC treatment (1 mM) for 24 h. NAC, N-acetyl-L-cysteine (<i>n</i> = 10). Values are means + SD, *p<0.05. (E) p-p38 MAPK and p-JNK levels in <i>Crif1</i>-deficient 3T3-L1 adipocytes with/without NAC treatment (1 mM) for 24 h. p38 MAPK, p38 mitogen-activated protein kinases; JNK, c-Jun N-terminal kinases. (F) Inhibition of <i>Mcp1</i> expression after treatment with p-p38 (SB203580; 10 µM for 24 h) or p-JNK (SP60125; 20 µM for 24 h) inhibitors, respectively (<i>n</i> = 10). Values are means + SD, *p<0.05. (G) Migration of Raw264.7 macrophages examined using culture supernatants from control siRNA- or <i>Crif1</i> siRNA-treated 3T3-L1 adipocytes with/without NAC (<i>n</i> = 10). Values are means + SD, *p<0.05.</p

Depletion of macrophages in adipose tissue by clodronate.

<p>The clodronate study was performed after feeding <i>Crif1^+/+,Fabp4</i> and <i>Crif1^f/+,Fabp4</i> mice a HFD for 8 weeks. Two intraperitoneal injections of clodronate were given with a 3 day interval between each. IPGTT and ITT were performed 6 days after the first injection. (A) Immunohistochemistry with anti-F4/80 after macrophage depletion by liposomal clodronate in <i>Crif1^+/+,Fabp4</i> and <i>Crif1^f/+,Fabp4</i> mice fed HFD. Scale: 200 µm. (B) Real-time PCR using primers for the macrophage marker, <i>Cd68</i> (<i>n</i> = 8). Values are means + SD, *p<0.05. (C and D) IPGTT and ITT after macrophage depletion by intraperitoneal injection of liposomal clodronate or PBS control (<i>n</i> = 8). Values are means + SD, *p<0.05, <i>Crif1^f/+,Fabp4</i> mice versus control mice; + p<0.05, clodronate versus PBS in control mice.</p

Marked failure of adipose tissue development in <i>Crif1^f/f,Fabp4</i> mice.

<p>Mice were generated by breeding two mouse lines, a <i>Crif1^flox/flox</i> transgenic mouse line and a Fabp4-<i>Cre</i> recombinase transgenic mouse line. Data from <i>Crif1^f/f,Fabp4</i> mice were obtained at 3 weeks-of-age. (A) Gross characteristics of control mice (<i>Crif1^+/+,Fabp4</i>), adipose-specific <i>Crif1</i> heterozygous mice (<i>Crif1^f/+,Fabp4</i>), and adipose-specific <i>Crif1</i> homozygous knockout mice (<i>Crif1^f/f,Fabp4</i>). TS, testis. (B and C) Body weight and tissue weight relative to control mice (<i>n</i> = 10). Values are means + SD, *p<0.05 versus control mice. BAT, brown adipose tissue; WAT, white adipose tissue. (D) <i>Crif1^f/f,Fabp4</i> mice showed markedly reduced survival rates after 2 weeks-of-age (<i>n</i> = 20). (E) Hematoxylin & eosin (H&E) staining of perirenal WAT (pWAT). Scale: 100 µm. (F) Transmission electron microscopy (TEM) of subcutaneous WAT (sWAT) revealed that the mitochondria of <i>Crif1^f/f,Fabp4</i> mice developed swollen cristae (red arrows). L, lipid droplet. Scale: 6,000 nm. (G) Mitochondria number per area in sWAT (<i>n</i> = 10). Values are means + SD. n.s, not significant.</p

Metabolic phenotypes and insulin resistance in <i>Crif1^f/+,Fabp4</i> mice.

<p>(A–D) An intraperitoneal glucose tolerance test (IPGTT) was performed with 1 g/kg glucose, after a 16 h fast in NCD or HFD mice (<i>n</i> = 8). Values are means ± SD, *p<0.05 versus control mice. (E) Western blot analysis of p-Akt in the gastrocnemius muscle and liver after injecting 1 U/kg insulin in control and <i>Crif1^f/+,Fabp4</i> mice with 14 weeks of HFD. Data are representative of three independent experiments. p-Akt, phospho-Akt; t-Akt, total-Akt. (F) Insulin (0.75 U/kg) tolerance tests (ITTs) (<i>n</i> = 8). Values are means ± SD, *p<0.05 versus control mice. (G) Hyperinsulinemic euglycemic clamp analysis in <i>Crif1^+/+,Fabp4</i> and <i>Crif1^f/+,Fabp4</i> mice with 14 weeks of HFD. HGP, hepatic glucose production (<i>n</i> = 8). Values are means + SD, *p<0.05, n.s, not significant.</p