High-Throughput Screening to Identify Plant Derived Human LDH-A Inhibitors

Abstract

Aims-Lactate dehydrogenase (LDH)-A is highly expressed in diverse human malignant tumors, parallel to aggressive metastatic disease, resistance to radiation/chemotherapy and clinically poor outcome. Although this enzyme constitutes a plausible target in treatment of advanced cancer, there are few known LDH-A inhibitors.Study Design-In this work, we utilized a high-throughput enzyme micro-array format to screen and evaluate > 900 commonly used medicinal plant extracts (0.00001-.5 mg/ml) for capacity to inhibit activity of recombinant full length human LDHA; EC .1.1.1.27.Methodology-The protein sequence of purified enzyme was confirmed using 1D gel electrophoresis-MALDI-TOF-MS/MS, enzyme activity was validated by oxidation of NADH (500μM) and kinetic inhibition established in the presence of a known inhibitor (Oxalic Acid). Conclusion-These findings reflect relative potency by rank of commonly used herbs and plants that contain human LDH-A inhibitory properties. Future research will be required to isolate chemical constituents within these plants responsible for LDH-A inhibition and investigate potential therapeutic application. Results-Of

1,2,3,4,6-Penta-O-galloylglucose within Galla Chinensis Inhibits Human LDH-A and Attenuates Cell Proliferation in MDA-MB-231 Breast Cancer Cells

“…High-throughput screening has identified Rhus chinensis gallnut ethanol extract with inhibitory activity on human recombinant hLDH-A. As previously reported, this was one of the most potent natural products elucidated with an IC 50 < 1 g/mL [18]. In order to elucidate the active constituents within the RCG extract responsible for hLDH-A inhibition, bioactivity guided fractionation (Figure 2(a)) was conducted to where hLDH-A activity was inhibited by the ethyl acetate fraction (167-215) (Figures 2(b), and 2(c)), which further yielded two active fractions within (Figure 2(d)).…”

“…A continuous hLDH-A assay using recombinant full length human LDHA (amino acids 1-332) with N terminal His tag, 352 amino acids with tag, and MW 38.8 kDa, enzyme commission (EC) number 1.1.1.27 (BRENDA | IUBMB) (Abcam, Cambridge, MA), was used, where we previously confirmed the identity of the enzyme Evidence-Based Complementary and Alternative Medicine 3 using matrix assisted laser desorption ionisation (MALDI) mass spectrometry and analysis by Mascot ID [18]. Briefly, the enzyme assay buffer consisted of HBSS + calcium and Mg, pH adjusted to 7.0.…”

“…Rhus chinensis gall (RCG) is the abnormal growth of Rhus chinensis leaves caused by Chinese aphid Schlechtendalia chinensis [8,9], also used for decades by indigenous people in the treatment of diarrhea, hemorrhage, and inflammation [10][11][12][13][14][15][16][17]. In a recent study conducted in our laboratory, a high-throughput screening of natural products revealed the extract of RGC to halt the proliferation of human breast cancer cells [7] and inhibit hLDH-A [18], an enzyme which plays a significant role in driving aggressive malignancies [2,[19][20][21], chemo-and radiotherapy resistance [22,23], tumorigenic potential [24][25][26], and metastatic processes [22]. It is believed that the abundance of lactic acid produced and released by tumor cells assists in invasion and motility [27] where a low pH weakens the extracellular stroma which helps tumor cells to dislodge and burrow in the blood and lymphatic systems [28].…”

Lactate dehydrogenase 5: An old friend and a new hope in the war on cancer

“…The ethanol extract of the Chinese gall, commonly named Wu Bei Zi, was found to be very effective within a therapeutic range [90]. However, the chemical component that is directly responsible for LDH-5 inhibition is currently unknown.…”

Stable shRNA Silencing of Lactate Dehydrogenase A (LDHA) in Human MDA-MB-231 Breast Cancer Cells Fails to Alter Lactic Acid Production, Glycolytic Activity, ATP or Survival

“…Previously, we conducted a high through put screening of natural products to identify human LDHA inhibitors, of which Chinese gallnut was a lead herb (17). Further analysis showed that 1,2,3,4,6-Penta-O-galloylglucose was the compound in Chinese gallnut with capacity to inhibit LDHA in the nM range (18).…”