Abstract
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. 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. 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). Of the natural extracts tested, the lowest IC50s [<0.001 mg/ml] were obtained by: Chinese Gallnut (Melaphis chinensis gallnut), Bladderwrack (Fucus vesiculosus), Kelp (Laminaria Japonica) and Babul (Acacia Arabica). Forty-six additional herbs contained significant LDH-A inhibitory properties with IC50s [<0.07 mg/ml], some of which have common names of Arjun, Pipsissewa, Cinnamon, Pink Rose Buds/Petals, Wintergreen, Cat's Claw, Witch Hazel Root and Rhodiola Root. 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.
Highlights
Human lactate dehydrogenase is a tetrameric enzyme [1] highly expressed in smooth muscle
A screening validation process was established using 0.02 U/ml LDH-A over 75 minutes (Fig. 1b) ± a known inhibitor; oxalic acid (Fig. 1c)
The enzyme used in this screening, was described as a recombinant full length Human LDHA with N terminal His tag; 352 amino acids with tag, MW 38.8 kDa: Enzyme Commission (EC) Number 1.1.1.27 (BRENDA | IUBMB) (Abcam, Cambridge, MA), we confirmed the identity of the enzyme using Matrix Assisted Laser Desorption Ionisation (MALDI) Mass Spec (MS/MS) and analysis by Mascot ID (Fig. 2)
Summary
Human lactate dehydrogenase is a tetrameric enzyme [1] highly expressed in smooth muscle. Unlike normal differentiated cells where lactate accumulation occurs anaerobically, cancer cells readily convert glucose into lactate aerobically, a phenomenon termed the Warburg effect [12]. Elevated protein expression or enzyme function of LDH–A is a contributor to accumulated lactate, and aggressive tumor growth [13], advanced progression [14], metastasis [15,16,17], acidity [18], and subsequent resistance to radiation and chemotherapy [19,20,21,22]. LDH–A knockdown, or lowering the functional capacity of LDH-A can suppress tumor growth and metastasis [23], indicating that this enzyme could serve as a novel targeted cancer therapy strategy. We elucidate LDH–A inhibitory effects of commonly used medicinal herbal extracts rom around the world
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