Two small molecule compounds, LLL12 and FLLL32, exhibit potent inhibitory activity on STAT3 in human rhabdomyosarcoma cells

Abstract

Abstract. Signal transducers and activators of transcription 3 (STAT3) signaling is persistently activated in many types of cancer cells, and represents a valid target for anticancer drug design. However, few reports have described the constitutive activation of STAT3 in human sarcoma cells. In this study, we demonstrate that the STAT3 signaling pathway is constitutively activated in human rhabodomyosarcoma cells (RH28, RH30, and RD2). We also investigated the inhibitory effects of two newly developed small molecules, LLL12 and FLLL32, on the STAT3 signaling pathway in human rhabodomyosarcoma cells. Both LLL12 and FLLL32 downregulated STAT3 constitutively and interleukin-6 (IL-6) stimulated phosphorylated STAT3 (p-STAT3). The inhibition of STAT3 via LLL12 and FLLL32 was confirmed by the inhibition of STAT3 DNA binding activity. The downstream targets of STAT3, cyclin D1, Bcl-xL, and survivin were also downregulated by LLL12 and FLLL 32 at both messenger RNA and protein levels. The potency of LLL12 and FLLL32 to inhibit proliferation/viability in human rhabodomyosarcoma cells (RH28, RH30, and RD2) was higher than that of the 5 previously reported Janus kinase 2 (JAK2)/ STAT3 inhibitors (LLL3, WP1066, Stattic, S3I-201, and AG490) and curcumin. Thus, in this study, we investigated the inhibitory effects of two STAT3 inhibitors, LLL12 and FLLL32, on the STAT3 signaling pathway in human rhabodomyosarcoma cells; we also demonstrated their higher potency in inhibiting proliferation on human rhabodomyosarcoma cells as compared to other five JAK2/STAT3 inhibitors and curcumin. IntroductionRhabdomyosarcoma, a highly malignant mesenchymal tumor, originating from immature striaed muscle, is the most common soft tissue sarcoma in children (1). Patients with rhabodomyosarcoma have a high relapse rate of 30% (1,2). Despite aggressive therapy, incorporating surgery, radiation therapy and dose-intensive chemotherapy, mortality rate of relapsing rhabodomyosarcoma is still 50-80% (2,3). Thus, development of higher potency and less toxicity anticancer drugs is pivotal to improve survival for patients with rhabdomyosarcoma.Signal transducers and activators of transcription 3 (STAT3) signaling is constitutively activated in various types of cancer cells, such as breast cancer, prostate cancer, leukemia and lymphomas (4). Activated STAT3 binds the promoters of the targeted genes to control cell growth and survival. Thus, inhibition of STAT3 signaling pathway seems to be an attractive strategy for anticancer drug design (5,6). For the past few years, several small molecules of the JAK2/STAT3 inhibitors, such as WP1066 (7-9), LLL3 (10) and AG490 (11-13), Stattic (14,15) and S3I-201 (16,17), have been reported with a wide range of antitumor effects. Although persistent activation of STAT3 has been detected in diverse cancer cell lines and tissue, only few reports have described elevated p-STAT3 in rhabdomyosarcoma.

STAT3 and its targeting inhibitors in osteosarcoma

“…138 LLL12 and FLLL32 have been shown to inhibit STAT3 directly and indirectly via IL-6-mediated phosphorylation in human rhabdomyosarcoma cells, OS cells and a murine model of OS. 139,140 LLL12 was shown to enhance the antiproliferative chemotherapeutic effect of doxorubicin against OS cell lines, and requires further investigation of its potential for combined drug therapy for OS treatment. 141 SC-1, a structural analogue of cancer drug, sorafenib, was shown to inhibit OS cell proliferation and tumour growth in vivo.…”

“…LLL12 inhibited STAT3 DNA binding and decreased the expression of STAT3 target genes implicated in oncogenesis, specifically survivin, cyclin D1 and Bcl‐2 138 . LLL12 and FLLL32 have been shown to inhibit STAT3 directly and indirectly via IL‐6‐mediated phosphorylation in human rhabdomyosarcoma cells, OS cells and a murine model of OS 139,140 . LLL12 was shown to enhance the anti‐proliferative chemotherapeutic effect of doxorubicin against OS cell lines, and requires further investigation of its potential for combined drug therapy for OS treatment 141 .…”

Antiparasitic activity of FLLL-32 against four Babesia species, B. bovis, B. bigemina, B. divergens and B. caballi, and one Theileria species, Theileria equi in vitro, and Babesia microti in mice

“…FLLL32 has exceptional biochemical properties, and in particular inhibits signal transducer and activator of transcription 3 (STAT3) phosphorylation, DNA-binding activity, and transactivation, and demonstrates significant growth suppressive activity in a variety of human cancer cells (Lin et al, 2010a;Bill et al, 2010b;Lin et al, 2010b;Fossey et al, 2011;Wei et al, 2011;Bill et al, 2012). Furthermore, FLLL32 can suppress IFNα and interleukin-6induced STAT3 phosphorylation (Lin et al, 2010a;Onimoe et al, 2012).…”

Mechanism insights of curcumin and its analogues in cancer: An update

“…FLLL32 is more potent than its parent molecule curcumin in its targeting because of the substitution of two hydrogen molecules on the focal carbon of curcumin with a spiro‐cyclohexyl ring. In human hepatocellular malignant growth cells, it restrained IL‐6‐prompted STAT3 phosphorylation in colorectal, glioblastoma, numerous myeloma, rhabdomyosarcoma, and liver disease cell lines (Anthwal et al, 2014; Chuan et al, 2013; Emerit, 1994; Lauren et al, 2009; Lin et al, 2009; Takeda et al, 2004) It displayed higher strength at repressing proliferation and STAT3 DNA binding than curcumin and other JAK/STAT3 inhibitors in human rhabdomyosarcoma cells along with loss of mRNA and protein expression of surviving (Wei et al, 2011). It is additionally answered to hinder the multiplication of OSA cell lines and appeared to advance apoptosis through caspase 3/7activation alongside PARP cleavage.…”