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Fulltext Modeling Melanoma In Vitro and In Vivo

Beaumont KA, Mohana-Kumaran N, Haass NK

Healthcare (Basel), 2013;2(1):27-46.
PMID: 27429258 DOI: 10.3390/healthcare2010027

The behavior of melanoma cells has traditionally been studied in vitro in two-dimensional cell culture with cells adhering to plastic dishes. However, in order to mimic the three-dimensional architecture of a melanoma, as well as its interactions with the tumor microenvironment, there has been the need for more physiologically relevant models. This has been achieved by designing 3D in vitro models of melanoma, such as melanoma spheroids embedded in extracellular matrix or organotypic skin reconstructs. In vivo melanoma models have typically relied on the growth of tumor xenografts in immunocompromised mice. Several genetically engineered mouse models have now been developed which allow the generation of spontaneous melanoma. Melanoma models have also been established in other species such as zebrafish, which are more conducive to imaging and high throughput studies. We will discuss these models as well as novel techniques that are relevant to the study of the molecular mechanisms underlying melanoma progression.
Fulltext Targeting the intrinsic apoptosis pathway as a strategy for melanoma therapy

Mohana-Kumaran N, Hill DS, Allen JD, Haass NK

Pigment Cell Melanoma Res, 2014 Jul;27(4):525-39.
PMID: 24655414 DOI: 10.1111/pcmr.12242

Melanoma drug resistance is often attributed to abrogation of the intrinsic apoptosis pathway. Targeting regulators of apoptosis is thus considered a promising approach to sensitizing melanomas to treatment. The development of small-molecule inhibitors that mimic natural antagonists of either antiapoptotic members of the BCL-2 family or the inhibitor of apoptosis proteins (IAPs), known as BH3- or SMAC-mimetics, respectively, are helping us to understand the mechanisms behind apoptotic resistance. Studies using BH3-mimetics indicate that the antiapoptotic BCL-2 protein MCL-1 and its antagonist NOXA are particularly important regulators of BCL-2 family signaling, while SMAC-mimetic studies show that both XIAP and the cIAPs must be targeted to effectively induce apoptosis of cancer cells. Although most solid tumors, including melanoma, are insensitive to these mimetic drugs as single agents, combinations with other therapeutics have yielded promising results, and tests combining them with BRAF-inhibitors, which have already revolutionized melanoma treatment, are a clear priority.
Fulltext Targeting the B-cell lymphoma 2 anti-apoptotic proteins for cervical cancer treatment

Abdul Rahman SF, Xiang Lian BS, Mohana-Kumaran N

Future Oncol, 2020 Oct;16(28):2235-2249.
PMID: 32715755 DOI: 10.2217/fon-2020-0389

The B-cell lymphoma 2 (BCL-2) anti-apoptotic proteins have become attractive therapeutic targets especially with the development of BH3-mimetics which selectively target these proteins. However, it is important to note that expression levels of the anti-apoptotic proteins and their relevance in inhibiting apoptosis varies between different cell lineages. This addiction to certain anti-apoptotic proteins for survival, can be determined with various techniques and targeted effectively with selective BH3-mimetics. Studies have highlighted that anti-apoptotic proteins BCL-XL and MCL-1 are crucial for cervical cancer cell survival. Co-targeting BCL-XL and MCL-1 with selective BH3-mimetics yielded promising results in cervical cancer cell lines. In this review, we focus on the expression levels of the anti-apoptotic proteins in cervical cancer tissues and how to possibly target them with BH3-mimetics.
Fulltext CTP synthase: the hissing of the cellular serpent

Thangadurai S, Bajgiran M, Manickam S, Mohana-Kumaran N, Azzam G

Histochem Cell Biol, 2022 Dec;158(6):517-534.
PMID: 35881195 DOI: 10.1007/s00418-022-02133-w

CTP biosynthesis is carried out by two pathways: salvage and de novo. CTPsyn catalyzes the latter. The study of CTPsyn activity in mammalian cells began in the 1970s, and various fascinating discoveries were made regarding the role of CTPsyn in cancer and development. However, its ability to fit into a cellular serpent-like structure, termed 'cytoophidia,' was only discovered a decade ago by three independent groups of scientists. Although the self-assembly of CTPsyn into a filamentous structure is evolutionarily conserved, the enzyme activity upon this self-assembly varies in different species. CTPsyn is required for cellular development and homeostasis. Changes in the expression of CTPsyn cause developmental changes in Drosophila melanogaster. A high level of CTPsyn activity and formation of cytoophidia are often observed in rapidly proliferating cells such as in stem and cancer cells. Meanwhile, the deficiency of CTPsyn causes severe immunodeficiency leading to immunocompromised diseases caused by bacteria, viruses, and parasites, making CTPsyn an attractive therapeutic target. Here, we provide an overview of the role of CTPsyn in cellular and disease perspectives along with its potential as a drug target.
Fulltext Rapid spheroid assays in a 3-dimensional cell culture chip

Teh JL, Abdul Rahman SF, Domnic G, Satiyasilan L, Chear NJY, Singh D, et al.

BMC Res Notes, 2021 Aug 13;14(1):310.
PMID: 34389056 DOI: 10.1186/s13104-021-05727-0

OBJECTIVE: The spheroid model provides a physiological platform to study cancer cell biology and drug sensitivity. Usage of bovine collagen I for spheroid assays is costly especially when experiments are conducted in 24-well plates, as high volume of bovine collagen I is needed. The aim of the study was to downsize spheroid assays to a microfluidic 3D cell culture chip and compare the growth, invasion and response to drug/compound of spheroids embedded in the 3D chip to spheroids embedded in 24-well plates.
RESULTS: Spheroids generated from nasopharyngeal carcinoma cell line HK-1 continuously grew and invaded into collagen matrix in a 24-well plate. Similar observations were noticed with spheroids embedded in the 3D chip. Large spheroids in both 24-well plate and the 3D chip disintegrated and invaded into the collagen matrix. Preliminary drug sensitivity assays showed that the growth and invasion of spheroids were inhibited when spheroids were treated with combination of cisplatin and paynantheine at high concentrations, in a 24-well plate. Comparable findings were obtained when spheroids were treated with the same drug combination in the 3D chip. Moving forward, spheroid assays could be performed in the 3D chip in a more high-throughput manner with minimal time and cost.
Single Agent and Synergistic Activity of Maritoclax with ABT-263 in Nasopharyngeal Carcinoma (NPC) Cell Lines

Xiang BLS, Kwok-Wai L, Soo-Beng AK, Mohana-Kumaran N

Trop Life Sci Res, 2020 Oct;31(3):1-13.
PMID: 33214852 DOI: 10.21315/tlsr2020.31.3.1

The BCL-2 anti-apoptotic proteins are over-expressed in many cancers and hence are attractive therapeutic targets. In this study, we tested the sensitivity of two Nasopharyngeal Carcinoma (NPC) cell lines HK1 and C666-1 to Maritoclax, which is reported to repress anti-apoptotic protein MCL-1 and BH3 mimetic ABT-263, which selectively inhibits anti-apoptotic proteins BCL-2, BCL-XL and BCL-w. We investigated the sensitisation of the NPC cell lines to these drugs using the SYBR Green I assay and 3D NPC spheroids. We report that Maritoclax repressed anti-apoptotic proteins MCL-1, BCL-2, and BCL-XL in a dose- and time-dependent manner and displayed a single agent activity in inhibiting cell proliferation of the NPC cell lines. Moreover, combination of Maritoclax and ABT-263 exhibited synergistic antiproliferative effect in the HK1 cells. Similar results were obtained in the 3D spheroids generated from the HK1 cells. More notably, 3D HK1 spheroids either treated with single agent Maritoclax or combination with ABT-263, over 10 days, did not develop resistance to the treatment rapidly. Collectively, the findings illustrate that Maritoclax as a single agent or combination with BH3 mimetics could be potentially useful as treatment strategies for the management of NPC.
Combinations of indole based alkaloids from Mitragyna speciosa (Kratom) and cisplatin inhibit cell proliferation and migration of nasopharyngeal carcinoma cell lines

Domnic G, Jeng-Yeou Chear N, Abdul Rahman SF, Ramanathan S, Lo KW, Singh D, et al.

J Ethnopharmacol, 2021 Oct 28;279:114391.
PMID: 34224811 DOI: 10.1016/j.jep.2021.114391

ETHNOPHARMACOLOGICAL RELEVANCE: Mitragyna speciosa (Korth.) or kratom is a medicinal plant indigenous to Southeast Asia. The leaf of M. speciosa is used as a remedy in pain management including cancer related pain, in a similar way as opioids and cannabis. Despite its well-known analgesic effect, there is a scarce of information on the cancer-suppressing potential of M. speciosa and its active constituents.
AIM OF THE STUDY: To assess the potential applicability of M. speciosa alkaloids (mitragynine, speciociliatine or paynantheine) as chemosensitizers for cisplatin in Nasopharyngeal carcinoma (NPC) cell lines.
MATERIALS AND METHODS: The cytotoxic effects of the extracts, fractions and compounds were determined by conducting in vitro cytotoxicity assays. Based on the cytotoxic screening, the alkaloid extract of M. speciosa exhibited potent inhibitory effect on the NPC cell line NPC/HK1, and therefore, was chosen for further fractionation and purification. NPC cell lines NPC/HK1 and C666-1 were treated with combinations of cisplatin and M. speciosa alkaloids combinations in 2D monolayer culture. The effect of cisplatin and mitragynine as a combination on cell migration was tested using in vitro wound healing and spheroid invasion assays.
RESULTS: In our bioassay guided isolation, both methanolic and alkaloid extracts showed mild to moderate cytotoxic effect against the NPC/HK1 cell line. Both NPC cell lines (NPC/HK1 and C666-1) were insensitive to single agent and combination treatments of the M. speciosa alkaloids. However, mitragynine and speciociliatine sensitized the NPC/HK1 and C666-1 cells to cisplatin at ~4- and >5-fold, respectively in 2D monolayer culture. The combination of mitragynine and cisplatin also significantly inhibited cell migration of the NPC cell lines. Similarly, the combination also of mitragynine and cisplatin inhibited growth and invasion of NPC/HK1 spheroids in a dose-dependent manner. In addition, the spheroids did not rapidly develop resistance to the drug combinations at higher concentrations over 10 days.
CONCLUSION: Our data indicate that both mitragynine and speciociliatine could be potential chemosensitizers for cisplatin. Further elucidation focusing on the drug mechanistic studies and in vivo studies are necessary to support delineate the therapeutic applicability of M. speciosa alkaloids for NPC treatment.
Fulltext Synergistic anti-proliferative effects of combination of ABT-263 and MCL-1 selective inhibitor A-1210477 on cervical cancer cell lines

Lian BSX, Yek AEH, Shuvas H, Abdul Rahman SF, Muniandy K, Mohana-Kumaran N

BMC Res Notes, 2018 Mar 27;11(1):197.
PMID: 29580266 DOI: 10.1186/s13104-018-3302-0

OBJECTIVE: There are number of studies which report that BCL-2 anti-apoptotic proteins (e.g. BCL-2, BCL-XL, and MCL-1) are highly expressed in cervical cancer tissues compared to the normal cervical epithelia. Despite these reports, targeting these proteins for cervical cancer treatment has not been explored extensively. BH3-mimetics that inhibit specific BCL-2 anti-apoptotic proteins may hold encouraging treatment outcomes for cervical cancer management. Hence, the aim of this pilot study is to investigate the sensitivity of cervical cancer cell lines to combination of two BH3-mimetics namely ABT-263 which selectively inhibits BCL-2, BCL-XL and BCL-w and A-1210477, a selective MCL-1 inhibitor.
RESULTS: We report that combination of A-1210477 and ABT-263 exhibited synergistic effects on all cervical cancer cell lines tested. Drug sensitization studies revealed that A-1210477 sensitised the cervical cancer cell lines SiHa and CaSki to ABT-263 by 11- and fivefold, respectively. Sensitization also occurred in the opposite direction whereby ABT-263 sensitised SiHa and CaSki to A-1210477 by eightfold. This report shows that combination of ABT-263 and A-1210477 could be a potential treatment strategy for cervical cancer. Extensive drug mechanistic studies and drug sensitivity studies in physiological models are necessary to unleash the prospect of this combination for cervical cancer therapy.
Fulltext Co-inhibition of BCL-XL and MCL-1 with selective BCL-2 family inhibitors enhances cytotoxicity of cervical cancer cell lines

Abdul Rahman SF, Muniandy K, Soo YK, Tiew EYH, Tan KX, Bates TE, et al.

Biochem Biophys Rep, 2020 Jul;22:100756.
PMID: 32346617 DOI: 10.1016/j.bbrep.2020.100756

Development of resistance to chemo- and radiotherapy in patients suffering from advanced cervical cancer narrows the therapeutic window for conventional therapies. Previously we reported that a combination of the selective BCL-2 family inhibitors ABT-263 and A-1210477 decreased cell proliferation in C33A, SiHa and CaSki human cervical cancer cell lines. As ABT-263 binds to both BCL-2 and BCL-XL with high affinity, it was unclear whether the synergism of the drug combination was driven either by singly inhibiting BCL-2 or BCL-XL, or inhibition of both. In this present study, we used the BCL-2 selective inhibitor ABT-199 and the BCL-XL selective inhibitor A1331852 to resolve the individual antitumor activities of ABT-263 into BCL-2 and BCL-XL dependent mechanisms. A-1210477 was substituted for the orally bioavailable S63845. Four cervical cancer cell lines were treated with the selective BCL-2 family inhibitors ABT-199, A1331852 and S63845 alone and in combination using 2-dimensional (2D) and 3-dimensional (3D) cell culture models. The SiHa, C33A and CaSki cell lines were resistant to single agent treatment of all three drugs, suggesting that none of the BCL-2 family of proteins mediate survival of the cells in isolation. HeLa cells were resistant to single agent treatment of ABT-199 and A1331852 but were sensitive to S63845 indicating that they depend on MCL-1 for survival. Co-inhibition of BCL-2 and MCL-1 with ABT-199 and S63845, inhibited cell proliferation of all cancer cell lines, except SiHa. However, the effect of the combination was not as pronounced as combination of A1331852 and S63845. Co-inhibition of BCL-XL and MCL-1 with A1331852 and S63845 significantly inhibited cell proliferation of all four cell lines. Similar data were obtained with 3-dimensional spheroid cell culture models generated from two cervical cancer cell lines in vitro. Treatment with a combination of A1331852 and S63845 resulted in inhibition of growth and invasion of the 3D spheroids. Collectively, our data demonstrate that the combination of MCL-1-selective inhibitors with either selective inhibitors of either BCL-XL or BCL-2 may be potentially useful as treatment strategies for the management of cervical cancer.
Modeling nasopharyngeal carcinoma in three dimensions

Siva Sankar P, Che Mat MF, Muniandy K, Xiang BLS, Ling PS, Hoe SLL, et al.

Oncol Lett, 2017 Apr;13(4):2034-2044.
PMID: 28454359 DOI: 10.3892/ol.2017.5697

Nasopharyngeal carcinoma (NPC) is a type of cancer endemic in Asia, including Malaysia, Southern China, Hong Kong and Taiwan. Treatment resistance, particularly in recurring cases, remains a challenge. Thus, studies to develop novel therapeutic agents are important. Potential therapeutic compounds may be effectively examined using two-dimensional (2D) cell culture models, three-dimensional (3D) spheroid models or in vivo animal models. The majority of drug assessments for cancers, including for NPC, are currently performed with 2D cell culture models. This model offers economical and high-throughput screening advantages. However, 2D cell culture models cannot recapitulate the architecture and the microenvironment of a tumor. In vivo models may recapitulate certain architectural and microenvironmental conditions of a tumor, however, these are not feasible for the screening of large numbers of compounds. By contrast, 3D spheroid models may be able to recapitulate a physiological microenvironment not observed in 2D cell culture models, in addition to avoiding the impediments of in vivo animal models. Thus, the 3D spheroid model offers a more representative model for the study of NPC growth, invasion and drug response, which may be cost-effective without forgoing quality.