Felicia Fei-Lei Chung

Toxicity of single-walled carbon nanotubes

Carbon nanotubes (CNTs) are an important class of nanomaterials, which have numerous novel properties that make them useful in technology and industry. Generally, there are two types of CNTs: single-walled nanotubes (SWNTs) and multi-walled nanotubes. SWNTs, in particular, possess unique electrical, mechanical, and thermal properties, allowing for a wide range of applications in various fields, including the electronic, computer, aerospace, and biomedical industries. However, the use of SWNTs has come under scrutiny, not only due to their peculiar nanotoxicological profile, but also due to the forecasted increase in SWNT production in the near future. As such, the risk of human exposure is likely to be increased substantially. Yet, our understanding of the toxicological risk of SWNTs in human biology remains limited. This review seeks to examine representative data on the nanotoxicity of SWNTs by first considering how SWNTs are absorbed, distributed, accumulated and excreted in a biological system, and how SWNTs induce organ-specific toxicity in the body. The contradictory findings of numerous studies with regards to the potential hazards of SWNT exposure are discussed in this review. The possible mechanisms and molecular pathways associated with SWNT nanotoxicity in target organs and specific cell types are presented. We hope that this review will stimulate further research into the fundamental aspects of CNTs, especially the biological interactions which arise due to the unique intrinsic characteristics of CNTs.

Metformin synergizes 5-fluorouracil, epirubicin, and cyclophosphamide (FEC) combination therapy through impairing intracellular ATP production and DNA repair in breast cancer stem cells

Metformin, an AMPK activator, has been reported to improve pathological response to chemotherapy in diabetic breast cancer patients. To date, its mechanism of action in cancer, especially in cancer stem cells (CSCs) have not been fully elucidated. In this study, we demonstrated that metformin, but not other AMPK activators (e.g. AICAR and A-769662), synergizes 5-fluouracil, epirubicin, and cyclophosphamide (FEC) combination chemotherapy in non-stem breast cancer cells and breast cancer stem cells. We show that this occurs through an AMPK-dependent mechanism in parental breast cancer cell lines. In contrast, the synergistic effects of metformin and FEC occurred in an AMPK-independent mechanism in breast CSCs. Further analyses revealed that metformin accelerated glucose consumption and lactate production more severely in the breast CSCs but the production of intracellular ATP was severely hampered, leading to a severe energy crisis and impairs the ability of CSCs to repair FEC-induced DNA damage. Indeed, addition of extracellular ATP completely abrogated the synergistic effects of metformin on FEC sensitivity in breast CSCs. In conclusion, our results suggest that metformin synergizes FEC sensitivity through distinct mechanism in parental breast cancer cell lines and CSCs, thus providing further evidence for the clinical relevance of metformin for the treatment of cancers.

Jerantinine A induces tumor-specific cell death through modulation of splicing factor 3b subunit 1 (SF3B1)

Precursor mRNA (pre-mRNA) splicing is catalyzed by a large ribonucleoprotein complex known as the spliceosome. Numerous studies have indicated that aberrant splicing patterns or mutations in spliceosome components, including the splicing factor 3b subunit 1 (SF3B1), are associated with hallmark cancer phenotypes. This has led to the identification and development of small molecules with spliceosome-modulating activity as potential anticancer agents. Jerantinine A (JA) is a novel indole alkaloid which displays potent anti-proliferative activities against human cancer cell lines by inhibiting tubulin polymerization and inducing G2/M cell cycle arrest. Using a combined pooled-genome wide shRNA library screen and global proteomic profiling, we showed that JA targets the spliceosome by up-regulating SF3B1 and SF3B3 protein in breast cancer cells. Notably, JA induced significant tumor-specific cell death and a significant increase in unspliced pre-mRNAs. In contrast, depletion of endogenous SF3B1 abrogated the apoptotic effects, but not the G2/M cell cycle arrest induced by JA. Further analyses showed that JA stabilizes endogenous SF3B1 protein in breast cancer cells and induced dissociation of the protein from the nucleosome complex. Together, these results demonstrate that JA exerts its antitumor activity by targeting SF3B1 and SF3B3 in addition to its reported targeting of tubulin polymerization.

Cudraflavone C induces tumor-specific apoptosis in colorectal cancer cells through inhibition of the phosphoinositide 3-kinase (PI3K)-AKT pathway

Cudraflavone C (Cud C) is a naturally-occurring flavonol with reported anti-proliferative activities. However, the mechanisms by which Cud C induced cytotoxicity have yet to be fully elucidated. Here, we investigated the effects of Cud C on cell proliferation, caspase activation andapoptosis induction in colorectal cancer cells (CRC). We show that Cud C inhibits cell proliferation in KM12, Caco-2, HT29, HCC2998, HCT116 and SW48 CRC but not in the non-transformed colorectal epithelial cells, CCD CoN 841. Cud C induces tumor-selective apoptosis via mitochondrial depolarization and activation of the intrinsic caspase pathway. Gene expression profiling by microarray analyses revealed that tumor suppressor genes EGR1, HUWE1 and SMG1 were significantly up-regulated while oncogenes such as MYB1, CCNB1 and GPX2 were down-regulated following treatment with Cud C. Further analyses using Connectivity Map revealed that Cud C induced a gene signature highly similar to that of protein synthesis inhibitors and phosphoinositide 3-kinase (PI3K)-AKT inhibitors, suggesting that Cud C might inhibit PI3K-AKT signaling. A luminescent cell free PI3K lipid kinase assay revealed that Cud C significantly inhibited p110β/p85α PI3K activity, followed by p120γ, p110δ/p85α, and p110α/p85α PI3K activities. The inhibition by Cud C on p110β/p85α PI3K activity was comparable to LY-294002, a known PI3K inhibitor. Cud C also inhibited phosphorylation of AKT independent of NFκB activity in CRC cells, while ectopic expression of myristoylated AKT completely abrogated the anti-proliferative effects, and apoptosis induced by Cud C in CRC. These findings demonstrate that Cud C induces tumor-selective cytotoxicity by targeting the PI3K-AKT pathway. These findings provide novel insights into the mechanism of action of Cud C, and indicate that Cud C further development of Cud C derivatives as potential therapeutic agents is warranted.

Cytochrome P450 2W1 (CYP2W1) in Colorectal Cancers

Cytochrome P450, family 2, subfamily W, polypeptide 1 (CYP2W1) is a newly identified monooxygenase enzyme that is expressed specifically in tumor tissues and during fetal life. Particularly, high expression of CYP2W1 was observed in up to 60% of colorectal cancers and its expression correlated with poor survival. CYP2W1 has been shown to metabolize various endogenous substrates including lysophospholipids and several procarcinogens, such as polycyclic aromatic hydrocarbon. The specific substrate for CYP2W1, however, is currently unknown. Due to its tumor-specific expression and its unique catalytic activities in colorectal cancers, CYP2W1 was deemed as an interesting target in colorectal cancer therapy. This review sought to summarize the current understanding of the CYP2W1 biology and biochemistry, its genetic polymorphisms and cancer risk, and its implication as a tumor-specific diagnostic and therapeutic target.

Contrasting sirtuin and poly(ADP-ribose)polymerase activities of selected 2,4,6-trisubstituted benzimidazoles

Both sirtuin and poly(ADP‐ribose)polymerase (PARP) family of enzymes utilize NAD+ as co‐substrate. Inhibitors of sirtuins and PARPs are important tools in drug discovery as they are reported to be linked to multiple diseases such as cancer. New potent sirtuin inhibitors (2,4,6‐trisubstituted benzimidazole) were discovered from reported PARP inhibitor scaffold. Interestingly, the synthesized compounds have contrasting sirtuin and PARP‐1 inhibitory activities. We showed that modification on benzimidazoles may alter their selectivity toward sirtuin or PARP‐1 enzymes. This offers an opportunity for further discovery and development of new promising sirtuin inhibitors. Molecular docking studies were carried out to aid the rationalization of these observations. Preliminary antiproliferative studies of selected compounds against nasopharyngeal cancer cells also showed relatively promising results.

Single-walled carbon nanotubes (SWCNTs) inhibit heat shock protein 90 (HSP90) signaling in human lung fibroblasts and keratinocytes

&NA; Single‐walled carbon nanotubes (SWCNTs) are carbon‐based nanomaterials that possess immense industrial potential. Despite accumulating evidence that exposure to SWCNTs might be toxic to humans, our understanding of the mechanisms for cellular toxicity of SWCNTs remain limited. Here, we demonstrated that acute exposure of short (1–3 &mgr;m) and regular‐length (5–30 &mgr;m) pristine, carboxylated or hydroxylated SWCNTs inhibited cell proliferation in human somatic and human stem cells in a cell type‐dependent manner. The toxicity of regular‐length pristine SWCNT was most evidenced in NP69 > CYT00086 > MCF‐10A > MRC‐5 > HaCaT > HEK‐293 T > HepG2. In contrast, the short pristine SWCNTs were relatively less toxic in most of the cells being tested, except for NP69 which is more sensitive to short pristine SWCNTs as compared to regular‐length pristine SWCNTs. Interestingly, carboxylation and hydroxylation of regular‐length SWCNTs, but not the short SWCNTs, significantly reduced the cytotoxicity. Exposure of SWCNTs also induced caspase 3 and 9 activities, mitochondrial membrane depolarization, and significant apoptosis and necrosis in MRC‐5 embryonic lung fibroblasts. In contrast, SWCNTs inhibited the proliferation of HaCaT human keratinocytes without inducing cell death. Further analyses by gene expression profiling and Connectivity Map analysis showed that SWCNTs induced a gene expression signature characteristic of heat shock protein 90 (HSP90) inhibition in MRC‐5 cells, suggesting that SWCNTs may inhibit the HSP90 signaling pathway. Indeed, exposure of MRC‐5 cells to SWCNTs results in a dose‐dependent decrease in HSP90 client proteins (AKT, CDK4 and BCL2) and a concomitant increase in HSP70 expression. In addition, SWCNTs also significantly inhibited HSP90‐dependent protein refolding. Finally, we showed that ectopic expression of HSP90, but not HSP40 or HSP70, completely abrogated the cytotoxic effects of SWCNTs, suggesting that SWCNT‐induced cellular toxicity is HSP90 dependent. In summary, our findings suggest that the toxic effects of SWCNTs are mediated through inhibition of HSP90 in human lung fibroblasts and keratinocytes. HighlightsSWCNTs inhibited cell proliferation in human somatic and human stem cells.SWCNTs induced cytotoxicity and growth inhibition in MRC‐5 and HaCat cells respectively.SWCNTs induced a gene signature characteristic of HSP90 inhibition.SWCNT‐induced toxicity in MRC‐5 and HaCat is HSP90‐dependent.

Abstract 2370: Depletion of p21-activated kinase 4, PAK4, inhibits cellular proliferation, motility and clonogenicity in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) accounts for 90% of all malignancies in the oral cavity, and is often associated with low survival and poor prognosis. While considerable progress in OSCC treatment has been made, options for targeted therapy remain limited. Thus, it is imperative that new druggable targets are identified and characterized. p21-activated kinase 4 (PAK4) is a member of the PAK family of serine/threonine kinases, which are known modulators of a diverse range of oncogenic pathways including cell proliferation, escape from apoptosis, and anchorage-independent growth. Previous reports have indicated that PAK4 is overexpressed in a subset of invasive OSCC, and is highly associated with poor prognosis. However, the specific role of PAK4 in OSCC has yet to be elucidated. Here, we show that RNAi-mediated depletion of PAK4 conferred reduced clonogenicity, cell motility, and cell proliferation in OSCC cell lines in a cell line-dependent manner. Global proteomic profiling of PAK4-depleted H103 and H357 cells was conducted to identify the molecules and signaling pathways responsive to PAK4 depletion. In addition to reported targets of PAK4 such as CFL1, ACTB, and TUBB6, we observed that p16INK4a, pre-mRNA splicing factors (SRSF2, SRSF5, SRSF6, SLU7), and proteasome activator subunits (PSME1-4) were dysregulated as a result of PAK4 depletion. These results indicate that PAK4 plays a role in promoting OSCC progression by regulating multiple signaling pathways. Of note, there have yet to be any reports describing interactions between PAK4 signaling and mRNA splicing or proteasome activation. Further investigation is thus warranted to further delineate the potential role of PAK4 in regulating these targets. Citation Format: Felicia F. Chung, Si-Hoey Tan, Vijay J. Raja, Pei Yuen Ng, Ian C. Paterson, Chye Ling Tan, Chee-Onn Leong. Depletion of p21-activated kinase 4, PAK4, inhibits cellular proliferation, motility and clonogenicity in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2370. doi:10.1158/1538-7445.AM2017-2370

Abstract 3506: Targeting breast cancer stem cells and non-stem breast cancer cells through combination therapies

Cancer stem cells (CSCs), as a subset of tumor cells with enhanced capacity to generate tumors, have recently been attributed to driving cancer recurrence and metastasis. Although the current CSC model highlights the importance of developing strategies to target CSCs, it is conceivable that the depletion of CSCs within a tumor would not lead to complete regression since non-CSCs might still be capable of sustaining tumor growth or regaining CSC potential. As either of these possibilities would confound the effectiveness of therapeutic agents that exclusively target CSCs, we aimed to rapidly identify combination therapies that could synergistically target both breast cancer cells and breast CSCs. Using a comprehensive small chemical library screen consisting of more than 1600 compounds, we identified a total of 193 small molecules, including 45 FDA approved drugs, which could target both breast CSCs and non-CSCs. When combined with conventional chemotherapeutic agents (e.g. cisplatin, doxorubicin or paclitaxel), histone deacetylase (HDAC) inhibitors exhibited significant synergistic effects in targeting both breast CSCs and non-CSCs. In summary, our data suggests that HDAC inhibitors represent a class of agents that could synergize with chemotherapeutic agents in refractory breast cancer cells and warrant further investigations. Citation Format: Ling-Wei Hii, Felicia Fei-Lei Chung, Boon-Shing Tan, Yang Mooi Lim, Soon-Keng Cheong, Chee-Onn Leong. Targeting breast cancer stem cells and non-stem breast cancer cells through combination therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3506. doi:10.1158/1538-7445.AM2015-3506