Loss-of-Function Genetic Screening Identifies Aldolase A as an Essential Driver for Liver Cancer Cell Growth Under Hypoxia.

BACKGROUND AND AIMS: Hypoxia is a common feature of the tumor microenvironment (TME), which promotes tumor progression, metastasis, and therapeutic drug resistance through a myriad of cell activities in tumor and stroma cells. While targeting hypoxic TME is emerging as a promising strategy for treating solid tumors, preclinical development of this approach is lacking in the study of HCC. APPROACH AND RESULTS: From a genome-wide CRISPR/CRISPR-associated 9 gene knockout screening, we identified aldolase A (ALDOA), a key enzyme in glycolysis and gluconeogenesis, as an essential driver for HCC cell growth under hypoxia. Knockdown of ALDOA in HCC cells leads to lactate depletion and consequently inhibits tumor growth. Supplementation with lactate partly rescues the inhibitory effects mediated by ALDOA knockdown. Upon hypoxia, ALDOA is induced by hypoxia-inducible factor-1α and fat mass and obesity-associated protein-mediated N6 -methyladenosine modification through transcriptional and posttranscriptional regulation, respectively. Analysis of The Cancer Genome Atlas shows that elevated levels of ALDOA are significantly correlated with poor prognosis of patients with HCC. In a screen of Food and Drug Administration-approved drugs based on structured hierarchical virtual platforms, we identified the sulfamonomethoxine derivative compound 5 (cpd-5) as a potential inhibitor to target ALDOA, evidenced by the antitumor activity of cpd-5 in preclinical patient-derived xenograft models of HCC. CONCLUSIONS: Our work identifies ALDOA as an essential driver for HCC cell growth under hypoxia, and we demonstrate that inhibition of ALDOA in the hypoxic TME is a promising therapeutic strategy for treating HCC.

Simultaneous high-performance liquid chromatographic determination of residual sulphamonomethoxine, sulphadimethoxine and their N4-acetyl metabolites in foods of animal origin.

A rapid and sensitive method for the determination of residual sulphamonomethoxine, sulphadimethoxine and their N4-acetyl metabolites in beef, pork, chicken and eggs by high-performance liquid chromatography (HPLC) was developed. The extraction of these compounds was performed using a mixture of 90% (v/v) acetonitrile solution and hexane (5:4, v/v) to minimize the fat content followed by purification by alumina column chromatography. These extracts contained sulphonamide analytes which were free from interfering compounds when examined by HPLC using a LiChrosorb RP-18 column. The average recoveries from spiked meat and egg were in excess of 80% with relative standard deviations between 0.4 and 5.0%. The practical limits of detection were 0.01 ppm for all samples.

Characterization of binding sites for sulfadimethoxine and its major metabolite, N4-acetylsulfadimethoxine, on human and rabbit serum albumin.

In order to gain an understanding of protein binding of sulfadimethoxine (SDM) and its major metabolite, N4-acetylsulfadimethoxine (N4-AcSDM), the binding of SDM and N4-AcSDM to human and rabbit serum albumin (HSA and RSA) was investigated using circular dichroism (CD), fluorescence and dialysis techniques. The CD spectral characteristics of the compounds bound to the albumins suggested that the drug-binding sites on the HSA and RSA had somewhat different asymmetries. The binding constants for SDM-HSA and -RSA interaction were smaller than those for N4-AcSDM. Two specific drug-binding sites were found on RSA, similarly to HSA, from the results of competitive displacement using fluorescence probes. Moreover, SDM and N4-AcSDM were found to share the same first binding site on the albumins. It can be presumed from the displacement data with a series of p-aminobenzoates that the characteristics of the binding sites (such as depth and width of the hydrophobic cleft) for SDM and N4-AcSDM on RSA may be almost the same, but the characteristics of these drug-binding sites on HSA may be somewhat different.

Role of deacetylation in the nonlinear pharmacokinetics of sulfamonomethoxine in pigs.

The role of deacetylation in the pharmacokinetics of sulfamonomethoxine (SMM) in pigs was studied using 6 Goettingen minipigs and a pig from a commercial breed. The rapid decrease of plasma concentration of the parent compound followed by the rapid increase of plasma concentration of the deacetylated metabolite, SMM, was observed after an i.v. injection of N4-acetylsulfamonomethoxine (AcSMM; 10 mg/kg). The concentration of the metabolite, SMM, was greater than that of AcSMM and after reaching peak, both compounds decreased in parallel on a semilogarithmic graph. On the other hand, the acetylated compound of SMM (AcSMM) appeared in the plasma and reached peak after an i.v. injection of SMM. After reaching the peak, both SMM and AcSMM decreased in parallel. The slopes of the terminal phases of SMM and AcSMM after both injections showed no significant difference. After the i.v. injection of a high dose of SMM (100 mg/kg), a nonlinear pharmacokinetics profile with capacity limited elimination type of SMM was observed. The concentration of plasma AcSMM increased rapidly and the parallel decrease of SMM and AcSMM was observed in both nonlinear and linear phases, while the renal excretion of AcSMM was saturated in the nonlinear phase. The results suggest that the deacetylation in pigs is so strong that it largely affected the pharmacokinetics of SMM in both high and low doses. In spite of the saturation in renal excretion of the main metabolite, AcSMM, after the high dose, the profile of the parallel decrease remained unchanged, which may be due to the rapid conversion of the excess AcSMM to SMM. The converted SMM was added to the existing plasma SMM. As a result, the nonlinear pharmacokinetics of SMM may have occurred after a high dose of SMM in pigs.

Contribution of renal active secretion in renal excretion of N4-acetylsulfamonomethoxine in conscious pigs.

The contribution of renal active tubular secretion of N4-acetylsulfamonomethoxine (AcSMM) in renal excretion was examined using 3 conscious pigs with permanent ureter cannulae. AcSMM was the main metabolite of sulfamonomethoxine (SMM) in pigs. When AcSMM (100 mg/kg) was intravenously injected, the renal clearance of AcSMM increased with the decrease of AcSMM plasma concentration. Michaelis-Menten kinetics was demonstrated in the renal excretion of AcSMM. It can be concluded that AcSMM is mostly excreted through active tubular secretion. The decrease of renal clearance of AcSMM may be thought as the causal factor of nonlinear plasma kinetics of SMM in pig.