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Chemotherapeutic drugs have been successfully used to treat several cancers, including melanoma. However, metastasis occasionally occurs after chemotherapy. Here, we reported that paclitaxel (PTX) treatment for B16F10 tumour in mice led to an enhanced lymphatic metastasis of the melanoma cells, although a significant inhibition of tumour growth at the injection site was observed. Further study demonstrated that PTX upregulated the expression of C-C chemokine receptor type 7 (CCR7) in B16F10 cells, enhancing their migration through the activation of JNK and p38 signalling pathways. Loss of CCR7 or blockade of C-C motif chemokine ligand 21 (CCL21)/CCR7 axis abolished the pro-migration effect of PTX on B16F10 melanoma cells. Importantly, combination of PTX and CCR7 mAb could simultaneously delay the tumour growth and reduce the lymphatic metastasis in B16F10 melanoma. The blockade of CCL21/CCR7 axis may collectively serve as a strategy for lymphatic metastasis in some melanoma after chemotherapy.

Cofactor regeneration is indispensable to avoid the addition of large quantities of cofactor NADH or NAD+ in oxidation-reduction reactions. Water-forming NADH oxidase (Nox) has attracted substantive attention as it can oxidize cytosolic NADH to NAD+ without concomitant accumulation of by-products. However, its applications have some limitations in some oxidation-reduction processes when its optimum pH is different from its coupled enzymes. In this study, to modify the optimum pH of BsNox, fifteen relevant candidates of site-directed mutations were selected based on surface charge rational design. As predicted, the substitution of this asparagine residue with an aspartic acid residue (N22D) or with a glutamic acid residue (N116E) shifts its pH optimum from 9.0 to 7.0. Subsequently, N20D/N116E combined mutant could not only downshift the pH optimum of BsNox but also significantly increase its specific activity, which was about 2.9-fold at pH 7.0, 2.2-fold at pH 8.0 and 1.2-fold at pH 9.0 that of the wild-type. The double mutant N20D/N116E displays a higher activity within a wide range of pH from 6 to 9, which is wider than the wide type. The usability of the BsNox and its variations for NAD+ regeneration in a neutral environment was demonstrated by coupling with a glutamate dehydrogenase for α-ketoglutaric acid (α-KG) production from L-glutamic acid (L-Glu) at pH 7.0. Employing the variation N20D/N116E as an NAD+ regeneration coenzyme could shorten the process duration; 90% of L-Glu were transformed into α-KG within 40 min vs. 70 min with the wild-type BsNox for NAD+ regeneration. The results obtained in this work suggest the promising properties of the BsNox variation N20D/N116E are competent in NAD+ regeneration applications under a neutral environment.