Investigation of Anti-Cancer Properties of Novel Curcuminoids in Leukemic Cells and Dalton Lymphoma Ascites Model

Abstract

Leukemia, one of the major causes of cancer death, ranks 11th worldwide among cancer-related deaths. The current treatment of leukemia faces challenges recently due to a high burden of side effects. It is well established that curcumin has anticancer and tumor-suppressing activities in several cancers in addition to leukemia. Accordingly, 15 derivatives were designed and prepared to improve the shortcomings of curcumin, such as poor aqueous solubility, chemical instability, and low bioavailability. All 15 were evaluated for cytotoxicity against the leukemic cell line MOLT-4, which led to the prioritization and further evaluation of compound curcuminoid (2E,5E)-2,5-bis((3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)cyclopentan-1-one 5i. 5i. Compared to curcumin, 5i was significantly more effective in inducing mitochondrial dysfunction in MOLT-4 cells; hence increased ROS production and cytotoxicity. Treatment groups showed change in mitochondrial membrane potential by flow cytometry analysis. Moreover, tumor volume reduction observed with 5i treatment in Dalton's Lymphoma model was accompanied with low toxicity. Intrinsic pathways of apoptosis was initiated by compound 5i that lowered Bcl-2 expression while augmenting cytochrome c, Bak and Bax levels both in vivo and in vitro. These results showcase the potent antiproliferative as well as cytotoxic effects of 5i at nanomolar doses against leukemia being at least 60 times more effective than curcumin.

Keywords: apoptosis; curcuminoids; cyclopentanone derivatives; cytotoxicity; leukaemia; monoketone.

Figure 1

Structures of curcumin, our synthesised curcuminoids, and curcuminoid 5i.

Scheme 1

Synthesis of curcuminoids 5a–o.

Figure 2

In vitro evaluation of 5i on cellular processes:(a) Cytotoxicity evaluation of 5i in MOLT-4 cell line using MTT assay. (b) Cytotoxicity evaluation of 5i in MOLT-4 cell line using resazurin assay. (c) Cell viability in HEK-293 cells upon the treatment of 5i. (d) Quantification of MMP depicted as a bar graph after 48 h treatment of 5i on MOLT-4 cells. (e) JC-1 staining indicating a change in mitochondrial membrane potential (MMP) upon 5i treatment. Every experiment was repeated three times and represented as histograms. p-value was calculated between control and 5i-treated groups. p-value representation: * (p-value ≤ 0.05), ** (p-value ≤ 0.01), *** (p-value ≤ 0.001), **** (p-value ≤ 0.0001).

Figure 3

Induction of apoptosis by 5i treatment:(a) Histograms and bar graph quantification of cell cycle assay of untreated and 5i-treated MOLT-4 cells for 48 h. (b) Dot plots and bar graph quantifications of apoptosis assay by AnnexinV-FITC/PI double staining of untreated and 5i-treated MOLT-4 cells for 48 h. (c) Western blot images of apoptotic protein markers in untreated and 5i-treated MOLT-4 cells for 48 h. I, II, and III represent control, 250 nM 5i, and 500 nM 5i, respectively. (d) Bar blots representing quantification of apoptotic protein markers in 5i-treated MOLT-4 cells. (e) Western blot images of apoptotic protein markers in DLA control and 5i-treated tumour tissues post 30 days of 5i treatment at a dosage of 50 mg/kg body weight. I represent untreated tumour and II represent 5i-treated tumour. (f) Quantification of apoptotic protein markers in DLA control and treated tumour. All bar graph data are represented as mean ± SD. All experiments were conducted three times. Significance was plotted based on p-value between treated tumour and its paired control condition. p-value representation: * (p-value ≤ 0.05), *** (p-value ≤ 0.001).

Figure 4

Histological and morphological analysis of the control and 5i-treated DLA mouse model: (a) Dot plot representing tumour volume for 30 days of treatment with 5i. Each dot represents the tumour volume of each individual animal. Diamond represents median tumour volume of untreated or treated for each time point. (b) Body weight measurements of control and treated animals (ns represents not significant). (c) Tumour, liver, spleen, and kidney of untreated and 5i-treated animals. (d) H&E staining of tumour, liver, and kidney after 30 days of 5i treatment.

Figure 5

Flowchart summarising experimental approach.