A Salmonella nanoparticle mimic overcomes multidrug resistance in tumours

Abstract

Salmonella enterica serotype Typhimurium is a food-borne pathogen that also selectively grows in tumours and functionally decreases P-glycoprotein (P-gp), a multidrug resistance transporter. Here we report that the Salmonella type III secretion effector, SipA, is responsible for P-gp modulation through a pathway involving caspase-3. Mimicking the ability of Salmonella to reverse multidrug resistance, we constructed a gold nanoparticle system packaged with a SipA corona, and found this bacterial mimic not only accumulates in tumours but also reduces P-gp at a SipA dose significantly lower than free SipA. Moreover, the Salmonella nanoparticle mimic suppresses tumour growth with a concomitant reduction in P-gp when used with an existing chemotherapeutic drug (that is, doxorubicin). On the basis of our finding that the SipA Salmonella effector is fundamental for functionally decreasing P-gp, we engineered a nanoparticle mimic that both overcomes multidrug resistance in cancer cells and increases tumour sensitivity to conventional chemotherapeutics.

Figure 1. The S. Typhimurium effector protein SipA modulates the expression of P-gp by an extracellular effect.

Western blot analysis showing P-gp expression in whole-cell lysates of HCT8 intestinal epithelial cell monolayers exposed to wild-type (WT) S. Typhimurium SL1344, SL1344 type III secretion system mutants, or wild-type SL1344-derived secreted protein extracts. GAPDH probing served as a loading control. Densitometries were analysed by ImageJ and presented as relative to the untreated cells; data represent the mean± s.d. of three independent experiments. (a) HCT8 intestinal epithelial cell monolayers were left untreated (−) or infected with WT SL1344 or SL1344 type III secretion system translocon mutant strains (ΔsipB or ΔsipC) for 5 h. (NS, not significant (Student’s t-test). (b) HCT8 cells were infected with wild type SL1344 or an SL1344 SPI-1-deficient mutant strain, or exposed to WT SL1344-derived secreted protein extracts for 5 h, and then probed as in a. P=0.0164 (Student’s t-test); significantly different compared with negative controls. (c) HCT8 cells were infected with WT SL1344, SL1344ΔsopA or ΔsipA, or SL1344ΔSipA complemented with a vector expressing SipA (ΔSipA/pSipA) for 5 h, and then probed as in a. *P=0.0007 (Student’s t-test).

Figure 2. SipA downregulates P-gp expression in a dose-dependent manner.

(a) HCT-8 cell monolayers were left untreated (−), or infected with wild-type SL1344, or exposed to 80 μg ml⁻¹, or 160 μg ml⁻¹ of purified SipA over a time course of 3 h. Normalized whole-cell lysates were then probed with P-gp and GAPDH. (b) HCT8 cell monolayers were infected with wild type SL1344 or exposed to purified lipopolysaccharide (LPS) from S. Typhimurium (0.1 to 100 μg ml⁻¹) for 3 h, and then probed as in a. (c) HCT8 cell monolayers were exposed to secreted protein extracts from SL1344 wild-type, ΔsipA or ΔSipA/pSipA for 3 h, and then probed as in a. Densitometry was analysed by ImageJ and presented as relative to the untreated cells; data represent the mean±s.d. of three independent experiments. (NS, not significant (Student’s t-test). All western blots were performed at least three times independently and the results shown are from one representative experiment.

Figure 3. S. Typhimurium modulates P-gp expression through a caspase-3-dependent mechanism.

(a) HCT8 cell monolayers were left untreated (−) or infected with wild-type SL1344 in the presence or absence of pharmacological inhibitors of CASP-3 or CASP-1 (negative control) for 5 h. Normalized whole-cell lysates were then probed with P-gp and GAPDH. The data represent a western blot analysis from an individual experiment performed at least three times. (b) HCT8 cell monolayers transfected with a nonspecific siRNA vector control or with siRNA aimed at decreasing CASP-3 expression were left untreated or infected with wild-type SL1344. Whole-cell lysates were then probed as in a. (c) Three-dimensional structure of mouse P-gp (PDB ID, 3G5U) depicted as cartoon and transparent surface. The cytoplasmic Caspase-3 cleavage site (⁴⁵⁴DGQD⁴⁵⁷) is shown in red. The putative CASP3 site ¹⁶⁴DVHD¹⁶⁷ is not shown. Numbers refer to the position of the amino acids in the protein sequence. (d) HCT8 cell monolayers were infected with wild type SL1344 for 1, 3 or 5 h, and then probed using a P-gp antibody capable of detecting P-gp cleavage products. Progressive P-gp modulation was accompanied by the occurrence of 90- and 60-kDa cleavage products. (e) Anexin-V staining was used as measurement of apoptosis in HCT8 cells 3 h post incubation with 160 or 320 μg ml⁻¹ of purified SipA. Staurosporine treatment served as the positive control. Shown is the average of three independent experiments with error bars indicating s.d.; *P=0.0076, **P=0.0009 (Student’s t-test).

Figure 4. SipA enhances the cytotoxic effect of doxorubicin on HCT8 cells.

(a) Alteration of the SipA CASP-3 motif did not alter the ability of S. Typhimurium to modulate P-gp. HCT8 cells were infected with wild-type SL1344, SL1344 ΔsipA or SL1344 ΔSipA complemented with a vector expressing SipA with a mutated CASP-3 cleavage motif (ΔSipA/pCSM-SipA) for 5 h. Whole-cell lysates were normalized for protein levels and probed for P-gp. GAPDH served as a loading control. The data represent a western blot analysis from an individual experiment performed at least three times. (b) HCT8 cells were treated with purified SipA (80 μg ml⁻¹), doxorubicin (5 μM) or vinblastine (5 μM) for 72 h. Cytotoxicity was measured using a colorimetric cell proliferation assay (see Methods section). (NS, not significant (Student’s t-test). Data were obtained from three independent experiments performed in triplicate and error bars indicate s.d. (c) Dose–response curves of doxorubicin and vinblastine (P-gp substrates), as well as 5-FU (not a P-gp substrate) in the absence (circle) or in the presence of purified-SipA (square). HCT8 cells were grown for 72 h with specific concentrations of the cytotoxic drugs (in μM) with or without purified-SipA (80 μg ml⁻¹). Cells viability was measure by CellTiter96 Aqueous One solution cell proliferation assay (see Methods section). Dose–response curves were derived from three independent experiments; error bars indicate±s.d. (n=3). Absent error bars indicate that error fell within the symbol. (d) Purified-SipA increases the intracellular accumulation of doxorubicin. HCT8 cells were treated with media only, doxorubicin, doxorubicin combined with purified-SipA, doxorubicin combined with purified-SifA or doxorubicin combined with verapamil. Accumulation of doxorubicin was evaluated by confocal microscopy as described in the Methods section. Red colour intensity represents intracellular doxorubicin accumulation. (e) The level of doxorubicin staining was quantified using the ImageJ software. Data represent the average of five readouts and error bars indicate s.d.; *P=0.001 (Student’s t-test).

Figure 5. The combined effect of SipA-AuNP delivery and exogenous doxorubicin treatment prevents tumour growth in a murine model of colon cancer.

(a) Schematic presentation of P-gp knockdown mechanism via SipA-AuNP. (b) SipA-AuNPs decrease the expression of P-gp at a SipA dose >100 times lower than free SipA. HCT8 cell monolayers were left untreated (−), exposed to 320 μg ml⁻¹ of purified SipA, AuNP alone or AuNP-SipA (0.72 μg ml⁻¹ of SipA) for 3 h. Whole-cell lysates were normalized for protein levels and probed for P-gp. GAPDH served as the loading control. All experiments were performed at least three times and the results shown are from one representative experiment. (c) The SipA-AuNPs conjugates improve the efficacy of doxorubicin. Balb/c mice bearing subcutaneous CT26 tumours (mean tumour volumes of ∼0.5 mm³) received i.p. treatments for 15 days (described in the Methods section) with AuNP alone (black square), SipA-AuNPs (green square), doxorubicin (blue triangle), AuNP in combination with doxorubicin (DOX) (black circle), SipA-AuNP in combination with DOX (black dots) or left untreated (red dot). Values indicate mean±s.d. (n=6); *P=0.0008, **P<0.0001 (one-way ANOVA). (d) P-gp expression in the tumours shown in c was evaluated by western blot. Tumours were homogenized and lysed, whole-cell lysates were normalized for protein levels and probed for P-gp. Levels of P-gp were quantified by densitometry and presented on the bar chart. Densitometry was performed using ImageJ and results are presented as relative to the untreated cells and error bars indicate s.d. (e) Accumulation of gold nanoparticles in the tumours shown in c was evaluated by SEM and X-ray microanalysis. Colour intensity represents tumour penetration. The sections of tumour were imaged for X-ray analysis and X-ray mapping as described in the Methods section. The cartoon inset depicts a schematic representation of the tumour architecture and basic components of cancer tissues. (f) BALB/c mice bearing subcutaneous CT26 tumours (mean tumour volumes of approximately 0.5 mm³) received i.p. treatments for 15 days as described in the Methods section. At necropsy, brain, spleen, heart, kidney, lung, liver and tumour tissues were collected. Tissue accumulation of SipA-AuNPs was determined by elemental analysis. Data represent the mean accumulation±s.d. (n=6). ANOVA, analysis of variance.

Figure 6. SipA-induced P-gp downregulation is conserved in other cancer cell types.

(a) MCF-7 breast adenocarcinoma cells were left untreated (−), or exposed to 80 μg ml⁻¹, or 160 μg ml⁻¹ of purified-SipA for 3 h. Normalized whole-cell lysates were then probed for P-gp and GAPDH. Densitometry was analysed by ImageJ and presented as relative to the untreated cells; data show the mean of three independent experiments±s.d. *P=0.0003; **P<0.0001 (Student’s t-test), significantly different compared with negative controls. (b) UM-UC-3 human bladder carcinoma cells were left untreated, or exposed to 80 μg ml⁻¹, or 160 μg ml⁻¹ of purified-SipA for 3 h, and then probed and analysed as in a; data show the mean of three independent experiments±s.d.*P<0.0001; **P<0.0001 (Student’s t-test), significantly different compared with negative controls. (c) SU10 diffuse large B-cell lymphoma cells were left untreated or exposed to 80, 160 or 320 μg ml⁻¹ of purified-SipA for 3 h, and then probed and analysed as in a; data show the mean of three independent experiments±s.d. *P<0.0001; **P=0.0078;***P<0.0001 (Student’s t-test), significantly different compared with negative controls (Student’s t-test). All experiments were performed at least three times and the results shown are from one representative experiment.

Figure 7. SipA-AuNP combined exogenous doxorubicin treatment prevents tumour growth in a NRG humanized model of primary breast cancer.

(a) Primary breast cancer tumour growth curves after 33 days of i.p. treatments (described in the Methods section) with AuNP alone (black square), SipA-AuNPs (green square), doxorubicin (blue triangle), AuNP in combination with DOX (black circle), SipA-AuNP in combination with DOX (black dot) or left untreated (red dot). Results represent mean±s.d. (n=3) tumour volume mm³; *P=0.0241, **P=0.0002 (one-way ANOVA). (b) Primary breast cancer tumours volumes at day 33 after i.p. treatments as mentioned above. Results represent mean±s.d.; (n of at least five mice per group). (c) P-gp expression in the tumours shown in a was evaluated by western blot. Tumours were homogenized and lysed, and whole-cell lysates were normalized for protein levels and probed for P-gp. Levels of P-gp were quantified by densitometry using ImageJ. Results are presented as relative to the untreated cells. Data represent means±s.d. *P=0.0171 (Student’s t-test). ANOVA, analysis of variance.

Figure 8. Working model of SipA downregulation of P-gp.

(a) Cancer cells express different types of ABC transporters, especially P-gp, to gain multidrug resistance. This allows tumour cells to extrude cytotoxic drugs from the intracellular space. (b) The SipA-AuNP may act extracellularly, by interacting with a transmembrane receptor to induce a CASP3 dependent cleavage of P-gp. The activation of caspase-3 also results in apoptosis; a cell death process. (c) Cleavage of P-gp results in the appearance of two protein fragments of about 90 and 60 kDa. Such cleavage destroys the P-go scaffold essentially removing this transporter from the plasma membrane thereby preventing the active efflux of doxorubicin and enhancing its cytotoxic activity. ABC, ATP-binding cassette.