In vitro and in vivo study on the anti-cancer effect of Hydrocotyle umbellata

다국어 초록 (Multilingual Abstract)

Background: Cancer is one of the most challenging diseases to treat and is characterized by abnormal and invasive growth and cellular metabolism reprogramming. To overcome these cancers, chemotherapy, surgery, and radiation therapy are the most common types of cancer treatments available today. However, these methods can cause immediate side effects and aftereffects of cancer treatment. Accordingly, research on cancer prevention and treatment using natural-derived substances to minimize side effects is being actively studied. Hydrocotyle umbellata is an aquatic plant belonging to Araliaceae and is known to have anti-anxiety, analgesic, and anti-inflammatory effects. However, the effect on cancer prevention and treatment is not well known. This study investigated the effect of H.umbellata on cancer growth and metabolism.
Methods: The leaves and stems of H.umbellata were freeze-dried after high pressure and high-temperature extraction. In vitro, the following experiment was conducted: cell growth measurement through cell counting, OCR and ECAR measurement through real-time cell metabolism assay, reactive oxygen species measurements, apoptosis, and measurement through metabolism-related gene through qRT-PCR analysis & western blot. The major components of H.umbellata were analyzed and separated through UPLC-PDA-QToF-MS. In vivo experiments, cancer cells and H.umbellata extract were injected into the mouse abdominal cavity or subcutaneous cavity to measure the weight of cancer masses.
Results: When H.umbellata's leaves and stem extracts were treated on cancer cells, growth decreased significantly, but the noncancerous cells, NIH3T3 cells (mouse fibroblast), had no noticeable growth difference. Based on these results, in vivo experiment was also conducted. This experiment confirmed that injecting B16F10 cells and H.umbellata extract into the mouse reduced cancer weight. The decrease in cancer cell proliferation is due to up-regulating expression level of PDK by H.umbellata extract, thereby reducing the expression of PDH, resulting in a decrease in ATP production due to oxidative phosphorylation occurring in mitochondria. ROS was also reduced due to mitochondrial dysfunction. The treatment of H.umbellata extract and 2-DG, glycolysis inhibitor, together on B16F10 cells showed more effective growth inhibition and cell death synergies than the treatment of the two substances alone. As a result, when analyzing the major components of H.umbellata, found 1,3,4-trihydroxy-2-butanyl-α-D-glucopyranoside and caffeoylquinic derivatives have anti-cancer effects.
Conclusions: This study suggests that H.umbellata regulates cell metabolism, causing mitochondrial dysfunction and inhibiting cancer cell growth.

목차 (Table of Contents)

• 1. Introduction 14
• 1-1 Risk of cancer and necessity of treatment 14
• 1-2 Various metabolic characteristics and direction of treatment of cancer cells 16
• 1-3 The necessity of combination treatment of glycolysis and OXPHOS inhibition 20
• 1-4 Natural plants for cancer therapy 22
• 1-5 Hydrocotyle umbellata and its pharmacological use 23
• 2. Material and Method 24
• 2-1 Preparation of H.umbellata extract 24
• 2-2 Cell culture 25
• 2-3 Measurement of cell growth 26
• 2-4 In vivo tumor model 27
• 2-5 Real-time cell metabolism assay 28
• 2-6 Reactive oxygen species measurements 29
• 2-7 Apoptosis assay 30
• 2-8 Quantitative real-time reverse-transcription-polymerase chain reaction (RT-PCR) analysis 31
• 2-9 Western blot analysis 32
• 2-10 UPLC-PDA-QToF-MS apparatus and chromatographic conditions 33
• 2-11 Statistical analysis 34
• 3. Results 35
• 3-1 H.umbellata extract suppresses cell proliferation selectively for cancer cells 35
• 3-2 Anti-tumor effects of H.umbellata extract in vivo 40
• 3-3 Inhibition of mitochondrial respiration of B16-F10 cells by H.umbellata extract 44
• 3-4 Evaluation of glycolysis of B16-F10 cells by H.umbellata extract 51
• 3-5 The combination of H.umbellata and 2-DG inhibited the proliferation of B16F10 cells significantly 54
• 3-6 Analysis of the major constituents of H.umbellata 57
• 4. Discussion 61
• 5. Reference 65