Modulation of angiogenesis by dithiolethione-modified NSAIDs and valproic acid

Abstract

Background and purpose: Angiogenesis involves multiple signaling pathways that must be considered when developing agents to modulate pathological angiogenesis. Because both cyclooxygenase inhibitors and dithioles have demonstrated anti-angiogenic properties, we investigated the activities of a new class of anti-inflammatory drugs containing dithiolethione moieties (S-NSAIDs) and S-valproate.

Experimental approach: Anti-angiogenic activities of S-NSAIDS, S-valproate, and the respective parent compounds were assessed using umbilical vein endothelial cells, muscle and tumor tissue explant angiogenesis assays, and developmental angiogenesis in Fli:EGFP transgenic zebrafish embryos.

Key results: Dithiolethione derivatives of diclofenac, valproate, and sulindac inhibited endothelial cell proliferation and induced Ser(78) phosphorylation of hsp27, a known molecular target of anti-angiogenic signaling. The parent drugs lacked this activity, but dithiolethiones were active at comparable concentrations. Although dithiolethiones can potentially release hydrogen sulphide, NaSH did not reproduce some activities of the S-NSAIDs, indicating that the dithioles regulate angiogenesis through mechanisms other than release of H(2)S. In contrast to the parent drugs, S-NSAIDs, S-valproate, NaSH, and dithiolethiones were potent inhibitors of angiogenic responses in muscle and HT29 tumor explants assessed by 3-dimensional collagen matrix assays. Dithiolethiones and valproic acid were also potent inhibitors of developmental angiogenesis in zebrafish embryos, but the S-NSAIDs, remarkably, lacked this activity.

Conclusions and implication: S-NSAIDs and S-valproate have potent anti-angiogenic activities mediated by their dithiole moieties. The novel properties of S-NSAIDs and S-valproate to inhibit pathological versus developmental angiogenesis suggest that these agents may have a role in cancer treatment.

Figure 1

(a) Corresponding amino-acid sequences of the S4 subunit of the voltage-gated Na_v1.4 (Popa et al., 2004) and K_v family (Shaker: K_v1.1 from Thornhill et al., 2003; K_v1.4 from http://vkcdb.biology.ualberta.ca/; K_v1.7 from Kalman et al., 1998; K_v1.8 (see Yao et al., 1995); Shab: K_v2.1 from Patel et al., 1997; Shaw: K_v3.1 and K_v3.4 from http://vkcdb.biology.ualberta.ca/) found in adult skeletal muscle (Gutman et al., 2005). Homologous sequences between Na_v domains and K_v are highlighted in grey. (b) Chemical structure of ajmaline.

Figure 2

(a and b), Images of the tip of the ‘loose-patch' pipette close to a muscle fibre. A sharp penetrating microelectrode (arrow in b) was used to continuously monitor resting membrane potential E_m.

Figure 3

Stability of Na⁺ current (I_Na) recordings and time course of ajmaline effect. For 10 min, a protocol to record I_Na traces was run every 30 s, showing stable I_Na amplitudes and I_Na–V relations. After addition of 100 μM ajmaline (arrow, at ∼600 s), current amplitudes quickly declined and inhibition was complete within ∼90 s. Seal resistance R_S: 2.2 MΩ, pipette resistance R_P: 324 kΩ, correction factor A=0.87, resting membrane potential E_m=−45 mV. Note that the pulse protocol (inset) and the potential values of the I_Na–V plots shown correspond to the tip potential V_p rather than intracellular membrane potential.

Figure 4

Time and voltage dependence of I_Na during a 10-min control period (a) and following addition of 100 μM ajmaline in another single fibre. I_Na–V plots are shown after conversion of the applied tip potential to intracellular membrane potentials as described in the Methods. Ajmaline affects both peak amplitudes (c) and (b) voltage dependence of I_Na.

Figure 5

Concentration dependence of I_Na and I_K blocking with the ‘loose-patch' clamp technique. (a) Recordings of I_Na and I_K before (left panel, R_S: 2.0 MΩ, R_P: 288 kΩ, A=0.87, E_m=−47 mV) and after the addition of 25 μM ajmaline (middle panel, R_S: 1.8MΩ, R_P: 288 kΩ, A=0.86). The right panel shows the corresponding I–V relations. (b) Concentration dependence of relative peak I_Na inhibition by ajmaline. (c) Concentration dependence of relative ‘steady-state' I_K inhibition by ajmaline. IC₅₀ values derived from Hill fits are indicated. Error Bars are s.e.m. with n single fibres (paired data).

Figure 6

Ajmaline effects on steady-state activation of I_Na. (a) m_∞ curve derived from the I_Na–V relation of the fibre shown in Figure 5a before (control: filled circles) and after the addition of 25 μM ajmaline (open circles). (b) Idealized m_∞ curves reconstructed from the mean m_0.5 and slope factors k of Boltzmann fits derived from recordings as shown in (a). (c) Summary of m_0.5 and k values for the different ajmaline concentrations (white or shaded bars, respectively) and their corresponding controls (black bars). ^*P<0.05. Error Bars: s.e.m.

Figure 7

Ajmaline effects on steady-state inactivation of I_Na. (a) Original recordings of I_Na from double-pulse protocols before and after the addition of 25 μM ajmaline (R_S: 1.8 MΩ, R_P: 290 kΩ, A=0.86, E_m=−45 mV). Note the different scaling of the ordinate in (a) and (b). The h_∞ curve was calculated as described in the Methods and fitted by a Boltzmann fit (right panel). Ajmaline shifted the h_∞ curve to the left and flattened its slope. (b) Idealized h_∞ curves for different ajmaline concentrations reconstructed from mean h_0.5 and k values as shown in (c). ^*P< 0.05. Error Bars: s.e.m.

Figure 8

Ajmaline effects on K⁺-currents (I_K). (a) I_K recorded with the 2-MVC technique in a representative single muscle fibre before (left) and after the application of 100 μM ajmaline (right). Test pulses ranged from −50  to +130 mV. (b) Control fibres showed approximately 20% inactivation of I_K under maintained depolarization, as judged from the steady-state to peak-I_K amplitude ratio, whereas ajmaline greatly reduced this amount of inactivation both for 100 μM (circles) and 25 μM (open triangles; filled triangles: corresponding controls). (c) After addition of 100 μM ajmaline, steady-state I_K amplitudes were reduced to ∼40% of controls, especially for larger depolarizations. The effect was less pronounced but significant for 25 μM (P<0.02, three out of four fibres). (d) Ajmaline (100 μM) significantly increased the time constants of K⁺ channel activation (τ_act.) compared to controls. Error Bars: s.e.m.

Figure 9

Ajmaline effects on action potentials. (a) Membrane potentials recorded with the current clamp technique in a representative single muscle fibre before (left) and after the addition of 10 μM ajmaline (middle). Current pulses were increased in 100 nA steps of ∼1.5 ms duration. The threshold was shifted 100 nA towards larger currents after ajmaline addition. The right panel shows the increase in the decay time constant with ajmaline concentration from some single fibres. (b) Repetitive action potentials did not produce any hyperexcitability (i.e., generation of action potentials in the post-train period) after 10 μM ajmaline. (c) Double-pulse stimulation of action potentials shows a normal reduction in action potential amplitude when the second action potentials is elicited shortly after the first under control conditions but almost no reaction after addition of 10 μM ajmaline.