Effects of plant extracts from family Zingiberaceae on CD34, Wilms’ Tumor 1, and Hippo signaling pathways in Leukemic stem cell line

Abstract

Leukemia is a hematopoietic disorder with frequent incidence and high mortality rate. Chemotherapy is an effective method for leukemia treatment. However, most chemotherapeutic drugs cause unpleasant side effects in cancer patients. Moreover, some patients fail to achieve complete remission and relapse because of uneliminated leukemic cells, known as leukemic stem cells (LSCs). These cells are more resistant to chemotherapeutic drugs than leukemic cells. CD34 is a protein marker in hematopoietic stem cells (HSCs) and LSCs. Although the function of this protein is still unknown, previous studies have indicated that CD34 is upregulated by activation of protein kinase C (PKC). Additionally, leukemia overexpressing Wilms’ tumor 1 (WT1) protein is involved in leukemogenesis. Previous studies have indicated that the Hippo signaling pathway is crucial for the regulation of organ growth and cell proliferation, with the YAP/TAZ proteins acting as protein mediators. Dysregulation of this pathway leads to cancer development. However, there is less research on this pathway in leukemia. Plants from the Zingiberaceae family have been utilized in traditional medicines and cuisines in Thailand. Many previous studies have demonstrated that plants from this family possess various biological activities. Many studies have indicated that unregulated inflammation is involved in the development and progression of cancer. Thus, suppressing unregulated inflammation is one strategy that could prevent cancer initiation. This study focuses on the cytotoxicity screening of plant extracts from the Zingiberaceae family in a KG-1a leukemic stem cell line, comparing their effects to other cells. Additionally, candidate plants and their active compounds were examined for cancer prevention properties. Finally, the effects of the candidate plant extracts and their active compounds on WT1, CD34, and the Hippo signaling pathways in LSCs were further investigated. The cytotoxic screening of 10 crude ethanolic extract plants by MTT assay across 5 cell lines, including KG-1a (leukemic stem cell), K562 (chronic myelocytic leukemia), MCF-7 (breast cancer cell), A549 (lung carcinoma cell), and HeLa (cervical carcinoma cell) was examined. Peripheral blood mononuclear cells (PBMCs) were used as a normal cell model to compare with cancer cells. The results indicated that crude ethanolic extracts from Curcuma longa, Curcuma zedoaria, and Zingiber officinale exhibited effective cytotoxicity against KG-1a cells. These extracts also showed significant cytotoxicity in K562, MCF-7, and HeLa cells, while demonstrating low cytotoxicity in PBMCs. The active compounds of C. longa and C. zedoaria are curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin), while shogaol and gingerol are active compounds of Z. officinale. The cancer prevention properties of candidate plant extracts and their active compounds were investigated using IL-2 and TNF-α ELISA kits, and nitric oxide (NO) assay. The results indicated that crude ethanolic extracts from these three plants and their active compounds showed cancer prevention properties, as they can decrease cytokine and NO levels. The inhibitory effect of crude ethanolic extracts from these three plants and their active compounds on CD34 and WT1 protein expressions in KG-1a cells was investigated by Western blotting. After KG-1a cells were treated with non-cytotoxic concentrations (IC20 values) of crude ethanolic extracts and their active compounds, the results showed that crude ethanolic extracts from these three plants and their active compounds could inhibit CD34 and WT1 protein expressions. Additionally, crude ethanolic extracts showed more effectiveness than their active compounds, especially in WT1 expression. Furthermore, Western blotting also revealed no expression of YAP protein in leukemic cell lines. While KG-1a cells exhibited a low level of TAZ protein expression. The effects of curcumin and shogaol on cell death and cell cycle in KG-1a cells were investigated by flow cytometry. The results revealed that curcumin and shogaol could arrest KG-1a cells at the G2/M phase with cytotoxic concentrations (IC50 values). Moreover, both active compounds induced cell death in a dose- and time-dependent manner. To explore the mechanisms of cell death induced by curcumin and shogaol in KG-1a cells, the cells were incubated with curcumin and shogaol at IC50 concentrations and the expression of apoptotic-related proteins were analyzed using Western blotting. The findings demonstrated that curcumin and shogaol induced cell death in KG-1a cells via apoptosis pathway by inhibiting the PI3K/Akt pathway, resulting in an upregulation of cleaved caspase-3 and PARP proteins. Additionally, curcumin and shogaol decreased the expression of WT1. However, no significant change was observed in TAZ after treatment, despite indications of reduction. In addition, the essential oils from 8 plants within the Zingiberaceae family were investigated for cytotoxicity in cancer and leukemic cell lines using MTT assay. The results indicated that the essential oil from Curcuma aeruginosa (CAEO) demonstrated the most effective cytotoxicity in K562 cells, whereas the essential oil from Zingiber ottensii (ZOEO) exhibited the highest cytotoxicity in MCF-7 cells. Subsequently, both essential oils were assessed for their impact on apoptosis, cell cycle arrest, and the cytotoxicity of essential oil-loaded nanoformulations in cell lines. Results from apoptosis and cell cycle analysis by flow cytometry revealed that CAEO arrested K562 cells at the G2/M phase and induced cell apoptosis in a dose-dependent manner. CAEO also exhibited anti-migration activity in MCF-7 cells. However, CAEO-loaded nanoemulsion formulations generally exhibited less cytotoxicity than CAEO alone, except for the nanoemulsion formulation against MCF-7 cells. On the other hand, ZOEO induced cell apoptosis in a dose-dependent manner but could not induce cell cycle arrest in MCF-7 cells. Moreover, formulated nanoemulsions and nanogels demonstrated significantly stronger cytotoxicity in A549, MCF-7, HeLa, and K562 cells than ZOEO alone. These findings indicate the potential of essential oils from plants within the Zingiberaceae family for leukemia and cancer treatment. In conclusion, these findings suggest that plant extracts from the Zingiberaceae family exhibit anti-cancer activities and cancer prevention properties. Furthermore, curcumin and shogaol, the main active compounds, demonstrated efficacy against leukemic stem cells. Thus, these two active compounds are suitable for study in the treatment of leukemia, which needs further investigation.