Cancer Stem Cells as a Prognostic Biomarker and Therapeutic Target Using Curcumin/ Piperine Extract for Multiple Myeloma

Abstract

Background: Multiple myeloma (MM) is a hematological bone marrow malignancy that can be treated but is usually fatal. Medication resistance is the major cause of relapses due to cancer stem cells (CSCs). As a result, this study aimed to identify multiple myeloma cancer stem cells (MMCSCs) in the bone marrow of twelve MM patients with pathological complete response (pCR) after chemotherapy and to investigate the potential effect of Curcumin/Piperine (C/P) extract as an anti-MMCSCs treatment in twenty newly diagnosed patients.

Methods: This study included twenty bone marrow (BM) samples from newly diagnosed MM patients and twelve BM samples from pCR patients after a year of treatment. The MTT test was performed to assess the treatment's effective dosage. A flow cytometer was used to identify MMCSCs, cell cycle profile, extract's apoptotic activity, and proliferation marker in the selected samples. Also, a colony formation test and stemness protein were investigated.

Results: In newly diagnosed MM patients, the C/P extract suppressed MMCSCs by 64.71% for CD138-/CD19- and 38.31% for CD38++. In MM patients' samples obtained after one year of treatment, the MMCSCs inhibition percentage reached 44.71% (P < 0.008) for CD138-/CD19- and 36.94% (P < 0.221) for CD38++. According to cell cycle analyses, the number of cells treated with C/P extract was significantly reduced in the S and G0/G1 phases (87.38%: 35.15%, and 4.83%: 2.17% respectively), with a rapid increase in the G2/M phases (1.1%: 2.2%.). MMCSCs apoptosis was identified using a flow cytometer and Annexin-V. Multiple myeloma stem cell (MMCSC) proliferation was inhibited. Clonogenicity was suppressed by 60%, and stemness protein expression was reduced by 70%.

Conclusion: MMCSCs in the bone marrow of MM-pCR patients can be utilized as a prognostic tool to predict recurrent multiple myeloma incidence. Also, the therapeutic potential of C/P extract as a prospective anti-MM drug targeting MMCSCs.

Keywords: Cancer stem cells; Curcumin/Piperine (C/P) extract; Multiple myeloma cancer stem cells; multiple myeloma; pathological complete response.

Figure 1

MTT Results to Detect the Viability of MM Cells after Treatment with C/P Extract

Figure 2

Identification of MMCSCs. (A, B) The cytotoxic effect of C/P extracts on the MMCSCs in comparison to control and chemotherapeutic groups using flow cytometry. Data were expressed as a median. (C) The ratio of CD19-/CD138- population was evaluated before and after treatment of C/P extract and chemotherapy. (D) The ratio of the CD38++ population was evaluated before and after treatment of C/P extract and chemotherapy

Figure 3

The Cell Cycle of MMCSCs Treated with C/P Extract Compared to Untreated Control

Figure 4

Apoptosis and Proliferation in MM Bone Marrow Samples Before and After C/P Extract Treatment (A) Annexin V and Propidium Iodide flow cytometric dot plot chart in untreated and treated MMCSCs. (B) Flow cytometric analysis was used to determine the percentage of apoptotic cells before and after treatment with the extract. When compared to untreated cells, data were reported as mean SEM (* P<0.05).

Figure 5

Under an Inverted Microscope, the Size and Number of the Colonies Reduced Following Treatment with the Extract

Figure 6

Morphological Apoptotic Changes in MM were induced by C/P extract and visualized at 1000X magnification. (A) Apoptotic body development in a plasma cell after treatment in a bone marrow smear aspirate. (B) A big binucleated leukemic blast cell with typical apoptotic bodies is shown with black arrows. (c) The black arrow indicates the discharge of apoptotic corpses from a damaged cell, whereas the red arrow indicates the creation of separate cytoplasmic or psedopods. (D) Apoptotic bodies are formed in giant plasma cells. (E) Apoptotic bodies develop in a plasma cell as shown by the arrow. (F) A blast cell's nuclear chromatin condenses along with the nuclear envelope, as shown by the red arrow

Figure 7

The Western Blotting Results for Stemness Protein in the Control Group, and C.P. Extract Group. (A) The membrane of a western blot for the stemness proteins (OCT-4A, NANOG, and SOX2) (C1, C2, C3: control for sample no. 1, control for sample no.2, control for sample no.3) (T1, T2, T3: test for sample no.1, test for sample no.1, test for sample no.1). (B-D) illustrate the effect of C/P extract on the levels of different proteins when compared with the control group