Plasmatic carbonic anhydrase IX as a diagnostic marker for clear cell renal cell carcinoma

Laura Lucarini, Lucia Magnelli, Nicola Schiavone, Alfonso Crisci, Alessio Innocenti, Luca Puccetti, Fabio Cianchi, Sara Peri, Claudiu T Supuran, Laura Papucci, Emanuela Masini, Laura Lucarini, Lucia Magnelli, Nicola Schiavone, Alfonso Crisci, Alessio Innocenti, Luca Puccetti, Fabio Cianchi, Sara Peri, Claudiu T Supuran, Laura Papucci, Emanuela Masini

Abstract

Carbonic anhydrase (CA, EC 4.2.1.1) IX is regarded as a tumour hypoxia marker and CA inhibitors have been proposed as a new class of antitumor agents, with one such agent in Phase II clinical trials. The expression of some CAs, in particular the isoforms CA IX and CA XII, has been correlated with tumour aggressiveness and progression in several cancers. The aim of this study was to evaluate the possibility that CA IX could represent a marker related to clear cell Renal Cell Carcinoma (ccRCC). Bcl-2 and Bax, and the activity of caspase-3, evaluated in tissue biopsies from patients, were congruent with resistance to apoptosis in ccRCCs with respect to healthy controls, respectively. In the same samples, the CA IX and pro-angiogenic factor VEGF expressions revealed that both these hypoxia responsive proteins were strongly increased in ccRCC with respect to controls. CA IX plasma concentration and CA activity were assessed in healthy volunteers and patients with benign kidney tumours and ccRCCs. CA IX expression levels were found strongly increased only in plasma from ccRCC subjects, whereas, CA activity was found similarly increased both in plasma from ccRCC and benign tumour patients, compared to healthy volunteers. These results show that the plasmatic level of CA IX, but not the CA total activity, can be considered a diagnostic marker of ccRCCs. Furthermore, as many reports exist relating CA IX inhibition to a better outcome to anticancer therapy in ccRCC, plasma levels of CA IX could be also predictive for response to therapy.

Keywords: Carbonic anhydrase; clear cell renal cell carcinoma; isoform IX; tumours.

Figures

Figure 1.
Figure 1.
CA IX expression in ccRCC samples. mRNA levels (panel A, left side) evaluated by RT-PCR and protein levels (panel B, left side) evaluated by WB of CA IX in normal tissue (Control) and tumour sample (tumour) of a representative patient. GAPDH housekeeping gene amplification products and tubulin housekeeping protein were used to normalise values from RT-PCR and western blot, respectively; in right side of panel A (RT-PCR) and B (WB) are reported the densitometric values of samples from eight patients. Differences were significant for p ≤ .005 with t-test.
Figure 2.
Figure 2.
Evaluation of VEGF in ccRCC samples. Protein levels evaluated by WB (panel A) or ELISA (panel B) of VEGF in normal tissue (Control) and tumour sample (tumour). In panel A, right side, are reported the densitometric values of samples of WB from eight patients (differences were significant for p ≤ .005 with t-test). In panel A, left side, WB of VEGF from a representative patient is reported. Tubulin housekeeping protein was used to normalise values in WB. VEGF concentration by ELISA from same patients (panel B, left and right side) is expressed as pg/µg. Differences were significant for p ≤ .005 with t-test.
Figure 3.
Figure 3.
Procaspase-3 expression and caspase-3 activity in ccRCC samples. Protein levels evaluated by WB (panel A, left side) of Procaspase-3 in normal tissue (Control) and tumour sample (tumour) of a representative patient. Tubulin housekeeping protein was used to normalise values in WB. In panel A, right side, are reported the densitometric values of samples from eight patients. Caspase-3 activity in samples was evaluated as reported in material and methods and data are reported in panel B, left and right side. Differences were significant for p ≤ .005 with t-test.
Figure 4.
Figure 4.
Ratio BAX/BCL2 in ccRCC samples. Protein levels of BCL2 and BAX evaluated by WB in normal tissue (Control) and tumour sample (tumour) of a representative patient. Tubulin was used to normalise values in WB. In left panel are reported the ratio between the values of BCL2 versus BAX. Differences were significant for p ≤ .005.
Figure 5.
Figure 5.
Evaluation of CA IX plasmatic concentration and CA enzymatic activity. Plasmatic concentration of CA IX evaluated by ELISA (upper panel). Plasmatic activity of CA (bottom panel) evaluated as reported in material and methods. Differences were significant for p ≤ .005 with t-test.

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